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Serum Cystatin C and the Risk of Coronary Heart Disease in Ethnic Chinese Patients With Normal Renal Function Rui Zhao, MMed, Yan Li, PhD,* Wen Dai, PhD Laboratory Medicine 47:1:13-19 DOI: 10.1093/labmed/lmv004
Background: Cystatin C, a marker for early stage chronic kidney dysfunction, mediates the pathogenesis of cardiovascular diseases. Objective: To investigate whether serum cystatin C is an independent predictor of coronary heart disease (CHD) for patients without chronic kidney disease (CKD). Methods: In this cross-sectional study, we included 525 patients with CHD and 142 apparently healthy control individuals and acquired their demographic characteristics, medical histories, and listings of the concurrent medications they were taking. All patients with CHD underwent coronary angiography and other routine laboratory examinations. We quantified serum concentrations of cystatin C via particle-enhanced immunonephelometric assay.
Results: Compared with controls, the patients with CHD had higher serum cystatin C levels (median: 1.17 mg/L, interquartile range [IQR]: 1.01 to 1.46 mg/L vs median: 0.98 mg/L, IQR: 0.80 to 1.06 mg/L, P 5.001), lower levels of high-density lipoprotein cholesterol (P5.001), higher levels of low-density lipoprotein cholesterol (P5.001), triglyceride (P5.001), total cholesterol (P ¼ .007) and high-sensitivity C-reactive protein (P5.001). No differences occurred in serum concentrations of creatinine or estimated glomerular filtration rate between CHD patients and healthy controls. Multivariate logistic regression indicated that levels of cystatin C (OR, 1.648; 95% confidence interval [CI], 1.454 to 1.868; P5.001) were most strongly correlated with CHD prevalence. Conclusions: Elevated serum cystatin C is a potential biochemical marker for the prediction of CHD, which suggests that mild preclinical renal dysfunction may be associated with the pathogenesis of CHD. Keywords: coronary heart disease, cystatin C, chronic kidney disease, glomerular filtration rate
Coronary heart disease (CHD) is characterized pathologically by atherosclerosis; the progression of atherosclerotic lesions is one of the key determinants in the prognosis of patients with CHD.1,2 Accumulating evidence3 suggests that complications of renal dysfunction may accelerate the progression of coronary atherosclerosis.3 Consequently,
patients with chronic renal failure are exposed to increased morbidity and mortality, partly due to the increased risk of major adverse cardiovascular events. Similarly, cardiovascular disease has become one of the most frequent complications of patients with renal disease.4 However, whether subclinical renal dysfunction played a role in the pathogenesis of CHD has not been fully determined.
Abbreviations
Several biomarkers or indices have been proposed as indicators of renal dysfunction. Glomerular filtration rate (GFR) is the most frequently used indicator of renal function. In routine clinical practice, GFR is usually not directly measured. Instead, it is estimated using the abbreviated Modification of Diet in Renal Disease (MDRD) equation based on the level of serum creatinine (SCr).5 However, study results6 have indicated that the levels of SCr and estimated GFR are substantially influenced by individual patient characteristics, such as muscle mass and weight, and that this index is less sensitive in patients with mild renal dysfunction.6 Cystatin C, a member of the human cystatin
CHD, coronary heart disease; GFR, glomerular filtration rate; MDRD, Modification of Diet in Renal Disease; SCr, serum creatinine; CKD, chronic kidney disease; CAG, coronary angiography; LAD, left anterior descending coronary artery; IQR, interquartile range; hs-CRP, high-sensitivity C-reactive protein; HDL-c, high-density lipoprotein cholesterol; TC, total cholesterol; TG, thyroglobulin; LDL-c, low-density lipoprotein cholesterol; BUN, blood urea nitrogen; UA, uric acid; OR, odds ratio; CI, confidence interval; NA, not applicable Clinical Laboratory Department, Renmin Hospital of Wuhan University, Wuhan, China *To whom correspondence should be addressed. [email protected]
C American Society for Clinical Pathology, 2015. All rights reserved. For permissions, please e-mail: [email protected] V
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ABSTRACT
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superfamily and a cysteine protease inhibitor involved in the catabolism of proteins, has been found to function as an early and sensitive marker of renal dysfunction.
Materials and Methods
Blood Specimen Collection and Laboratory Analysis We collected blood specimens before CAG from the antecubital veins of the patients, who were resting in supine position and had been fasting for at least 8 hours. All standardized laboratory analyses were performed according to manufacturer’s instructions. Serum levels of cystatin C were measured via particle-enhanced immunonephelometric assay using the Siemens ADVIA 2400 autoanalyzer (Siemens AG, Munich, Germany). We also evaluated routine laboratory parameters using the Siemens ADVIA 2400 autoanalyzer. Serum creatine (SCr) was determined using an enzymatic method. GFR was estimated using the MDRD equation, using coefficients corrected for the ethnic Chinese population based on the concentration of SCr [GFR (mLmin11.73 m2) ¼ 175 SCr1.154 Age0.203 ( 0.742, if female)]. A patient with a GFR of greater than or equal to 60 mLmin11.73 m2 was considered to not have CKD, which was consistent with the findings of a previous study.22
Patients and Healthy Subject Individuals Statistical Analysis We enrolled 525 patients with CHD (patient group) who were hospitalized at Renmin Hospital, Wuhan University, China, and 142 apparently healthy volunteers. All patients with CHD underwent coronary angiography (CAG). Reductions in coronary-artery lumen diameter and the roentgenographic appearance of concentric lesions and eccentric plaques were evaluated (reductions of 25%, 50%, 75%, 90%, and 99%). Each principal vascular segment was assigned a multiplier in accordance with the functional significance of the myocardial area supplied by that segment: the left main coronary artery, the left anterior descending coronary artery (LAD), the proximal segment of the circumflex artery, the mid-segment of the LAD, the right coronary artery, the distal segment of the LAD, the posterolateral artery, the obtuse
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Normally distributed values of laboratory parameters were presented as mean (SD). Skewed data are expressed as median and interquartile range (IQR). Independent t testing and v2 testing were used for the comparison of continuous and categorical variables, respectively. If the data were not normally distributed, the Mann-Whitney U-test was used. Correlations between continuous variables were assessed using the Spearman correlation analysis. We performed logistic regression analysis to evaluate the relationship between the incidence of CHD and the parameters. All P values were 2-sided; P5.05 was considered significant. Statistical analyses were performed using SPSS Software, version 19.0 (SPSS Inc, Chicago, IL).
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Cystatin C is a low-molecular protein (13.4 kDa) that is synthesized at a constant rate in all nucleated cells. Cystatin C is freely filtered through the glomerulus and then reabsorbed and fully catabolized, without secretion or subsequent reabsorption into the circulation.7-9 Serum cystatin C is less likely to be influenced by individual characteristics, such as age, sex, ethnicity, muscle mass, and medication use, compared with SCr.10-12 Thus, researchers have proposed cystatin C as a more reliable endogenous marker of early renal dysfunction. Results of previous studies13,14 have suggested that serum cystatin C may be an independent risk factor for all-cause and cardiovascular mortality among elderly persons. It has also been reported15-18 that higher serum cystatin C may be associated with increased risk of cardiovascular disease and subsequent mortality without established chronic kidney disease (CKD), although the results were not always consistent.19 Studies from ethnic Chinese populations have investigated the association between increased serum cystatin C and the risk of CHD, particularly in individuals without apparent CKD. Therefore, the aim of our study was to evaluate whether serum cystatin C level is an independent predictor of CHD risk in patients without CKD.
marginal artery, and others.20 Only patients who were found to have 30% or greater stenosis of at least 1 major coronary artery during CAG were defined as having CHD.21 Exclusion criteria were patients with comorbidities including peripheral blood diseases, autoimmune diseases, cancer, and infectious and inflammatory diseases. We obtained written informed consent from all participants. All subjects underwent physical examination on enrollment in the study.
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Results Characteristics of the Study Population
hs-CRP, GFR, Cystatin C, and Other Biochemical Parameters vs Markers in Patients With CHD Subsequently, we performed a correlation analysis to evaluate whether other commonly used biochemical markers correlated with the values of hs-CRP, GFR, and cystatin C in patients with CHD. The results showed that the individual levels of GFR were significantly and negatively correlated with serum levels of blood urea nitrogen (BUN), SCr, uric acid (UA), and LDL-c for patients with CHD (P5.05 for all; Table 2; Figures 2A-2D). The hs-CRP levels were negatively correlated with the levels of HDL-c (P ¼ 0.002) but with no significant correlation with GFR (P> .05; Table 2 and Figure 2E). More importantly, our results showed a significant positive correlation between serum cystatin C and the conventional inflammatory marker hs-CRP (P5.001; Table 2 and Figure 2F).
Table 1. Clinical Characteristics of the Included Controls and Patients With Coronary Heart Disease Variable
Controls (n ¼ 142)
Patients With CHD (n ¼ 525)
Age (y), mean (SD) Sex (male/female) TC (mmol/L), mean (SD) TG (mmol/L), median (IQR) HDL-c (mmol/L), median (IQR) LDL-c (mmol/L), median (IQR) ALT (U/L), median (IQR) AST (U/L), median (IQR) T-bil (mmol/L), median (IQR) BUN (mmol/L), mean (SD) SCr (mg/dl), median (IQR) UA (mmol/L), median (IQR) GFR (mlmin1 1.73 m2), median (IQR) Cys C (mg/L), median (IQR) hs-CRP (mg/L), median (IQR) Treatment, % Statin use Aspirin use Antihypertensive therapy
60.54 (7.98) 106/36 3.94 (0.85) 1.27 (.96-1.85) 1.07 (0.98-1.25) 2.52 (2.02-2.87) 23.0 (18.00-30.00) 23.0 (20.00-28.00) 10.71 (8.16-14.59) 5.56 (1.17) 0.87 (0.76-1.02) 318.0 (285.0-354.0) 88.36 (74.89-109.50) 0.98 (0.80-1.06) 1.25 (0.63-2.66)
60.69 (8.58) 384/141 4.18 (0.96) 1.70 (1.27-2.31) 0.97 (0.84-1.13) 2.76 (2.25-3.20) 22.0 (15.00-32.00) 24.0 (18.00-33.00) 11.29 (8.46-14.42) 5.49 (1.41) 0.89 (0.71-1.03) 325.0 (270.0-383.0) 87.54 (76.17-106.87) 1.17 (1.01-1.46) 1.91 (0.86-6.83)
16.90 13.60 19.72
21.52 16.67 23.05
P Valuea .85 .75 .007 5.001 5.001 5.001 .71 .38 .28 .53 .68 .40 .72 5.001 5.001 .24 .58 .43
TC, total cholesterol; TG, triglycerides; IQR, interquartile range; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; T-bil, total bilirubin; BUN, blood urine nitrogen; SCr, serum creatinine; UA, uric acid; GFR, glomerular filtration rate; Cys C, cystatin C; hsCRP, high-sensitivity C-reactive protein; NA, not applicable. a Bolding indicates statistical significance.
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The clinical characteristics of the patients with CHD patients and the controls are listed in Table 1. Serum cystatin C levels were significantly higher in the patients with CHD compared to controls (Figure 1A; median: 1.17 mg/L, IQR: 1.01 to 1.46 mg/L vs median: 0.98 mg/L, IQR: 0.80 to 1.06 mg/L; P5.001). No significant differences, however, were detected in serum SCr or GFR between the patients with CHD and controls. High-sensitivity C-reactive protein (hs-CRP) levels were higher in the patients with CHD compared with the controls (Figure 1B; median: 1.91 mg/L, IQR: 0.86-6.83 mg/L vs median: 1.25 mg/L, IQR: 0.63 to 2.66 mg/L; P5.001). The high-density lipoprotein cholesterol (HDL-c) concentration was lower in the patients with CHD than in the controls (median: 0.97 mmol/L, IQR: 0.84 to 1.13 mmol/L vs median: 1.07 mmol/L, IQR: 0.98 to 1.25 mmol/L; P5.001), whereas serum total cholesterol (TC), thyroglobulin (TG), and low-density lipoprotein cholestrole (LDL-c) were higher (mean [SD], 4.18 [0.96] mmol/L vs 3.94 [0.85] mmol/L, P ¼ .007; median: 1.70 mmol/L, IQR: 1.27 to
2.31 mmol/L vs median 1.27 mmol/L, IQR: 0.96 to 1.85 mmol/ L, P5.001; median: 2.76 mmol/L, IQR: 2.25 to 3.20 mmol/L vs median: 2.52 mmol/L, IQR: 2.02 to 2.87 mmol/L, P5.001, respectively) in patients with CHD.
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Figure 1
Table 2. Correlations Within the Serum Levels of Various Analytes and Lipid Profiles in Patients With CHDa Analyte hs-CRP Spearman correlation P value GFR Spearman correlation P value Cys C Spearman correlation P value
GFR
hs-CRP
BUN
SCr
UA
TC
TG
HDL-c
LDL-c
NA NA
NA NA
0.091 .04
0.034 .44
0.008 .86
0.018 .68
0.138 .002
0.137 .002
0.151 .001
NA NA
0.057 .19
0.305 5.001
0.850 5.001
0.321 5.001
0.013 .77
0.106 .02
0.071 .10
0.132 .002
0.075 .09
0.420 5.001
0.017 .70
0.076 .08
0.039 .37
0.017 .70
0.103 .02
0.132 .002
0.066 .13
CHD, coronary heart disease; GFR, glomerular filtration rate; hs-CRP, high-sensitivity C-reactive protein; BUN, blood urine nitrogen; SCr, serum creatinine; UA, uric acid; TC, total cholesterol; TG, triglyceride; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; Cys C, cystatin C. a Bolding indicates statistical significance.
Logistic Regression Analysis of Independent Predictors of CHD Prevalence To assess the factors predicting the prevalent risk of CHD, we performed univariate and multivariate logistic regression analyses. Results of the univariate analysis showed that serum levels of TC, TG, HDL-c, LDL-c, hs-CRP, and cystatin C were associated with the prevalence of CHD (Table 3). Further, the results of multivariate analysis indicated that the level of cystatin C (OR ¼ 1.648, 95% confidence interval [CI], 1.454 to 1.868, P5.001; Table 3) was the strongest independent predictor of CHD risk in the studied cohort.
Discussion CKD has been recognized as a significant burden for patients with CHD. In patients with CHD, comorbidities of
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CKD further increased the risk of incidence of major adverse cardiovascular events and death. Currently, the diagnosis of CKD is based on GFR values, which are calculated primarily from serum concentrations of SCr. However, SCr has been found to be affected by many factors other than renal function, such as age, sex, muscle mass, and physicalactivity levels.6,10-12 In fact, patients with a mild decline in renal function, GFR evaluation based on SCr level might be biased. A study23 indicated that cystatin C, a protein that belongs to the family of cysteine proteinase inhibitors and is synthesized in a stable manner by almost all nucleated cells, may better reflect renal function.23 Further, cystatin C is a highly sensitive marker for renal function, reflecting preclinical renal dysfunction when SCr levels and GFR remain normal. Elevated serum cystatin C levels are related to increased risk of CHD24 and severity of coronary atherosclerotic lesions,25,26 particularly in patients with CKD. However, the association of cystatin C and CHD risk in patients without CKD has not been fully investigated. 27
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Serum levels of analytes in control subjects and patients with coronary heart disease (CHD). A, Cystatin C levels. B, High-sensitivity C-reactive protein (hs-CRP). The values were presented as medians and the interquartile ranges because these data were not normally distributed.
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Scatter diagrams of comparative data from our cohort. These diagrams illustrate the correlation between glomular filtration rate (GFR) and levels of blood urea nitrogen (BUN) (A), serum creatinine (SCr) (B), uric acid (UA) (C), low-density lipoprotein cholesterol (LDL-c) (D), and serum cystatin C (E); and between levels of serum cystatin C and high-sensitivity C-reactive protein (hs-CRP) (F).
Table 3. Univariate and Multivariate Logistic Regression Analysis of Factors Predicting CHD Prevalencea Variable
Age Sex TC TG HDL-c LDL-c ALT AST T-bil BUN SCr UA GFR(mlmin1 1.73 m2) Cys C hs-CRP
Univariate
Multivariate
OR (95% CI)
P Value
OR (95% CI)
P Value
1.002 (.980-1.024) 1.227 (.787-1.913) 1.328 (1.078-1.635) 1.922 (1.466-2.521) 0.170 (.078-.370) 1.993 (1.503-2.643) 1.008 (0.996-1.021) 1.013 (0.999-1.027) 1.028 (0.990-1.067) 0.957 (0.835-1.096) 0.946 (0.373-2.396) 1.001 (0.999-1.004) 1.000 (0.992-1.007) 1.703 (1.508-1.922) 1.154 (1.087-1.226)
.85 .37 .008 5.001 5.001 5.001 .21 .06 .15 .53 .91 .29 .94 5.001 5.001
NA NA 0.888 (0.634-1.245) 1.472 (1.065-2.035) 0.371 (0.136-1.013) 1.749 (1.109-2.758) NA NA NA NA NA NA NA 1.648 (1.454-1.868) 1.031 (0.968-1.097)
NA NA .49 .02 .05 .02 NA NA NA NA NA NA NA 5.001 .34
OR, odds ratio; CI, confidence interval; CHD, coronary heart disease; TC, total cholesterol; TG, triglyceride; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; T-bil, total bilirubin; BUN, blood urine nitrogen; SCr, serum creatinine; UA, uric acid; GFR, glomerular filtration rate; Cys C, cystatin C; hs-CRP, high-sensitivity C-reactive protein. a Bolding indicates statistical significance.
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Figure 2
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Despite these findings, it is currently unclear whether cystatin C is a direct marker of cardiovascular diseases or only a marker of preclinical renal dysfunction in our cohort. Inflammation plays an important role in the pathogenesis and prognosis of CHD. The results of our study and other studies29,30 have suggested a close correlation between cystatin C and the conventional inflammatory maker hs-CRP. Therefore, it is reasonable to suggest that the cystatin C level may be of predictive value in CHD by reflecting the amount of systemic inflammation. Indeed, examination of the relationship between serum cystatin C and frequent inflammatory response in patients with declined renal function has been reported in the literature.31 However, a possible explanation for our results is that serum cystatin C is a more sensitive marker of early renal dysfunction, which may be involved in the process of CHD. Despite the uncertainty of the exact mechanisms underlying the predictive role of cystatin C in CHD, evidence suggests that elevated serum cystatin C is associated with worse prognosis in patients with CHD. A study by Zethelius et al32 assessed whether a combination of biomarkers, including cystatin C, N-terminal pro-brain natriuretic peptide, troponin, and hs-CRP, improved patient risk stratification compared with established cardiovascular risk factors. Those researchers discovered that adding cystatin C to the system significantly improved predictive efficacy.32 More direct evidence may be derived from animal studies or pathologic studies in humans, which have demonstrated that the cystatin C levels were decreased in tissues with atherosclerotic plaques and aneurysms.33,34 Also, a recent study report by Niccoli et al35 revealed that the concentration of blood cystatin C was directly proportional to the number of
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stenotic lesions in 70 patients with acute coronary syndrome and that cystatin C was associated with severity of coronary atherosclerosis plaque formation. Further studies are needed to confirm the precise mechanisms underlying the predictive and therapeutic value of cystatin C in CHD. Moreover, cystatin C seems to offer more complete prognostic information than other markers of renal disease and is one of the strongest risk predictors of CHD. Plasma cystatin C is a marker for CKD, a disease strongly associated with an increased risk for cardiovascular disease.36,37 Because of the association of renal dysfunction with cardiovascular disease, it is unclear whether cystatin C is a direct marker of CHD or merely a marker for renal dysfunction, which has implications for therapeutic intervention. Cystatin C is measured via particle-enhanced immunonephelometric assay, using particles coated with cystatin C–specific antibodies, and subsequent turbidimetry or nephelometry.38 The assays are precise: detection methods provide coefficients of variation ranging from 2% to 8%.39 Otherwise, the intra- and interindividual biologic variations of serum cystatin C are 5% and 13%, respectively.40 Our data showed that the serum cystatin C concentrations of patients with CHD were elevated by more than 20% compared with controls. These data could be used clinically and could imply clinical significance. The results of our study indicate that elevated serum cystatin C level is significantly associated with the risk prevalence for CHD in patients without CKD, suggesting that even subtle renal dysfunction, which cannot be detected via SCr levels, is associated with the incidence of CHD. Because the presence of CHD is one of the major determinants of poor prognosis in patients with impaired renal function, serum levels of cystatin C are expected to be useful in identifying patients at risk for CHD, even in the absence of CKD. LM
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Serum Cystatin C and the Risk of Coronary Heart Disease in Ethnic Chinese Patients With Normal Renal Function Rui Zhao, MMed, Yan Li, PhD,* Wen Dai, PhD Laboratory Medicine 47:1:13-19 DOI: 10.1093/labmed/lmv004
Background: Cystatin C, a marker for early stage chronic kidney dysfunction, mediates the pathogenesis of cardiovascular diseases. Objective: To investigate whether serum cystatin C is an independent predictor of coronary heart disease (CHD) for patients without chronic kidney disease (CKD). Methods: In this cross-sectional study, we included 525 patients with CHD and 142 apparently healthy control individuals and acquired their demographic characteristics, medical histories, and listings of the concurrent medications they were taking. All patients with CHD underwent coronary angiography and other routine laboratory examinations. We quantified serum concentrations of cystatin C via particle-enhanced immunonephelometric assay.
Results: Compared with controls, the patients with CHD had higher serum cystatin C levels (median: 1.17 mg/L, interquartile range [IQR]: 1.01 to 1.46 mg/L vs median: 0.98 mg/L, IQR: 0.80 to 1.06 mg/L, P 5.001), lower levels of high-density lipoprotein cholesterol (P5.001), higher levels of low-density lipoprotein cholesterol (P5.001), triglyceride (P5.001), total cholesterol (P ¼ .007) and high-sensitivity C-reactive protein (P5.001). No differences occurred in serum concentrations of creatinine or estimated glomerular filtration rate between CHD patients and healthy controls. Multivariate logistic regression indicated that levels of cystatin C (OR, 1.648; 95% confidence interval [CI], 1.454 to 1.868; P5.001) were most strongly correlated with CHD prevalence. Conclusions: Elevated serum cystatin C is a potential biochemical marker for the prediction of CHD, which suggests that mild preclinical renal dysfunction may be associated with the pathogenesis of CHD. Keywords: coronary heart disease, cystatin C, chronic kidney disease, glomerular filtration rate
Coronary heart disease (CHD) is characterized pathologically by atherosclerosis; the progression of atherosclerotic lesions is one of the key determinants in the prognosis of patients with CHD.1,2 Accumulating evidence3 suggests that complications of renal dysfunction may accelerate the progression of coronary atherosclerosis.3 Consequently,
patients with chronic renal failure are exposed to increased morbidity and mortality, partly due to the increased risk of major adverse cardiovascular events. Similarly, cardiovascular disease has become one of the most frequent complications of patients with renal disease.4 However, whether subclinical renal dysfunction played a role in the pathogenesis of CHD has not been fully determined.
Abbreviations
Several biomarkers or indices have been proposed as indicators of renal dysfunction. Glomerular filtration rate (GFR) is the most frequently used indicator of renal function. In routine clinical practice, GFR is usually not directly measured. Instead, it is estimated using the abbreviated Modification of Diet in Renal Disease (MDRD) equation based on the level of serum creatinine (SCr).5 However, study results6 have indicated that the levels of SCr and estimated GFR are substantially influenced by individual patient characteristics, such as muscle mass and weight, and that this index is less sensitive in patients with mild renal dysfunction.6 Cystatin C, a member of the human cystatin
CHD, coronary heart disease; GFR, glomerular filtration rate; MDRD, Modification of Diet in Renal Disease; SCr, serum creatinine; CKD, chronic kidney disease; CAG, coronary angiography; LAD, left anterior descending coronary artery; IQR, interquartile range; hs-CRP, high-sensitivity C-reactive protein; HDL-c, high-density lipoprotein cholesterol; TC, total cholesterol; TG, thyroglobulin; LDL-c, low-density lipoprotein cholesterol; BUN, blood urea nitrogen; UA, uric acid; OR, odds ratio; CI, confidence interval; NA, not applicable Clinical Laboratory Department, Renmin Hospital of Wuhan University, Wuhan, China *To whom correspondence should be addressed. [email protected]
C American Society for Clinical Pathology, 2015. All rights reserved. For permissions, please e-mail: [email protected] V
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ABSTRACT
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superfamily and a cysteine protease inhibitor involved in the catabolism of proteins, has been found to function as an early and sensitive marker of renal dysfunction.
Materials and Methods
Blood Specimen Collection and Laboratory Analysis We collected blood specimens before CAG from the antecubital veins of the patients, who were resting in supine position and had been fasting for at least 8 hours. All standardized laboratory analyses were performed according to manufacturer’s instructions. Serum levels of cystatin C were measured via particle-enhanced immunonephelometric assay using the Siemens ADVIA 2400 autoanalyzer (Siemens AG, Munich, Germany). We also evaluated routine laboratory parameters using the Siemens ADVIA 2400 autoanalyzer. Serum creatine (SCr) was determined using an enzymatic method. GFR was estimated using the MDRD equation, using coefficients corrected for the ethnic Chinese population based on the concentration of SCr [GFR (mLmin11.73 m2) ¼ 175 SCr1.154 Age0.203 ( 0.742, if female)]. A patient with a GFR of greater than or equal to 60 mLmin11.73 m2 was considered to not have CKD, which was consistent with the findings of a previous study.22
Patients and Healthy Subject Individuals Statistical Analysis We enrolled 525 patients with CHD (patient group) who were hospitalized at Renmin Hospital, Wuhan University, China, and 142 apparently healthy volunteers. All patients with CHD underwent coronary angiography (CAG). Reductions in coronary-artery lumen diameter and the roentgenographic appearance of concentric lesions and eccentric plaques were evaluated (reductions of 25%, 50%, 75%, 90%, and 99%). Each principal vascular segment was assigned a multiplier in accordance with the functional significance of the myocardial area supplied by that segment: the left main coronary artery, the left anterior descending coronary artery (LAD), the proximal segment of the circumflex artery, the mid-segment of the LAD, the right coronary artery, the distal segment of the LAD, the posterolateral artery, the obtuse
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Normally distributed values of laboratory parameters were presented as mean (SD). Skewed data are expressed as median and interquartile range (IQR). Independent t testing and v2 testing were used for the comparison of continuous and categorical variables, respectively. If the data were not normally distributed, the Mann-Whitney U-test was used. Correlations between continuous variables were assessed using the Spearman correlation analysis. We performed logistic regression analysis to evaluate the relationship between the incidence of CHD and the parameters. All P values were 2-sided; P5.05 was considered significant. Statistical analyses were performed using SPSS Software, version 19.0 (SPSS Inc, Chicago, IL).
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Cystatin C is a low-molecular protein (13.4 kDa) that is synthesized at a constant rate in all nucleated cells. Cystatin C is freely filtered through the glomerulus and then reabsorbed and fully catabolized, without secretion or subsequent reabsorption into the circulation.7-9 Serum cystatin C is less likely to be influenced by individual characteristics, such as age, sex, ethnicity, muscle mass, and medication use, compared with SCr.10-12 Thus, researchers have proposed cystatin C as a more reliable endogenous marker of early renal dysfunction. Results of previous studies13,14 have suggested that serum cystatin C may be an independent risk factor for all-cause and cardiovascular mortality among elderly persons. It has also been reported15-18 that higher serum cystatin C may be associated with increased risk of cardiovascular disease and subsequent mortality without established chronic kidney disease (CKD), although the results were not always consistent.19 Studies from ethnic Chinese populations have investigated the association between increased serum cystatin C and the risk of CHD, particularly in individuals without apparent CKD. Therefore, the aim of our study was to evaluate whether serum cystatin C level is an independent predictor of CHD risk in patients without CKD.
marginal artery, and others.20 Only patients who were found to have 30% or greater stenosis of at least 1 major coronary artery during CAG were defined as having CHD.21 Exclusion criteria were patients with comorbidities including peripheral blood diseases, autoimmune diseases, cancer, and infectious and inflammatory diseases. We obtained written informed consent from all participants. All subjects underwent physical examination on enrollment in the study.
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Results Characteristics of the Study Population
hs-CRP, GFR, Cystatin C, and Other Biochemical Parameters vs Markers in Patients With CHD Subsequently, we performed a correlation analysis to evaluate whether other commonly used biochemical markers correlated with the values of hs-CRP, GFR, and cystatin C in patients with CHD. The results showed that the individual levels of GFR were significantly and negatively correlated with serum levels of blood urea nitrogen (BUN), SCr, uric acid (UA), and LDL-c for patients with CHD (P5.05 for all; Table 2; Figures 2A-2D). The hs-CRP levels were negatively correlated with the levels of HDL-c (P ¼ 0.002) but with no significant correlation with GFR (P> .05; Table 2 and Figure 2E). More importantly, our results showed a significant positive correlation between serum cystatin C and the conventional inflammatory marker hs-CRP (P5.001; Table 2 and Figure 2F).
Table 1. Clinical Characteristics of the Included Controls and Patients With Coronary Heart Disease Variable
Controls (n ¼ 142)
Patients With CHD (n ¼ 525)
Age (y), mean (SD) Sex (male/female) TC (mmol/L), mean (SD) TG (mmol/L), median (IQR) HDL-c (mmol/L), median (IQR) LDL-c (mmol/L), median (IQR) ALT (U/L), median (IQR) AST (U/L), median (IQR) T-bil (mmol/L), median (IQR) BUN (mmol/L), mean (SD) SCr (mg/dl), median (IQR) UA (mmol/L), median (IQR) GFR (mlmin1 1.73 m2), median (IQR) Cys C (mg/L), median (IQR) hs-CRP (mg/L), median (IQR) Treatment, % Statin use Aspirin use Antihypertensive therapy
60.54 (7.98) 106/36 3.94 (0.85) 1.27 (.96-1.85) 1.07 (0.98-1.25) 2.52 (2.02-2.87) 23.0 (18.00-30.00) 23.0 (20.00-28.00) 10.71 (8.16-14.59) 5.56 (1.17) 0.87 (0.76-1.02) 318.0 (285.0-354.0) 88.36 (74.89-109.50) 0.98 (0.80-1.06) 1.25 (0.63-2.66)
60.69 (8.58) 384/141 4.18 (0.96) 1.70 (1.27-2.31) 0.97 (0.84-1.13) 2.76 (2.25-3.20) 22.0 (15.00-32.00) 24.0 (18.00-33.00) 11.29 (8.46-14.42) 5.49 (1.41) 0.89 (0.71-1.03) 325.0 (270.0-383.0) 87.54 (76.17-106.87) 1.17 (1.01-1.46) 1.91 (0.86-6.83)
16.90 13.60 19.72
21.52 16.67 23.05
P Valuea .85 .75 .007 5.001 5.001 5.001 .71 .38 .28 .53 .68 .40 .72 5.001 5.001 .24 .58 .43
TC, total cholesterol; TG, triglycerides; IQR, interquartile range; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; T-bil, total bilirubin; BUN, blood urine nitrogen; SCr, serum creatinine; UA, uric acid; GFR, glomerular filtration rate; Cys C, cystatin C; hsCRP, high-sensitivity C-reactive protein; NA, not applicable. a Bolding indicates statistical significance.
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The clinical characteristics of the patients with CHD patients and the controls are listed in Table 1. Serum cystatin C levels were significantly higher in the patients with CHD compared to controls (Figure 1A; median: 1.17 mg/L, IQR: 1.01 to 1.46 mg/L vs median: 0.98 mg/L, IQR: 0.80 to 1.06 mg/L; P5.001). No significant differences, however, were detected in serum SCr or GFR between the patients with CHD and controls. High-sensitivity C-reactive protein (hs-CRP) levels were higher in the patients with CHD compared with the controls (Figure 1B; median: 1.91 mg/L, IQR: 0.86-6.83 mg/L vs median: 1.25 mg/L, IQR: 0.63 to 2.66 mg/L; P5.001). The high-density lipoprotein cholesterol (HDL-c) concentration was lower in the patients with CHD than in the controls (median: 0.97 mmol/L, IQR: 0.84 to 1.13 mmol/L vs median: 1.07 mmol/L, IQR: 0.98 to 1.25 mmol/L; P5.001), whereas serum total cholesterol (TC), thyroglobulin (TG), and low-density lipoprotein cholestrole (LDL-c) were higher (mean [SD], 4.18 [0.96] mmol/L vs 3.94 [0.85] mmol/L, P ¼ .007; median: 1.70 mmol/L, IQR: 1.27 to
2.31 mmol/L vs median 1.27 mmol/L, IQR: 0.96 to 1.85 mmol/ L, P5.001; median: 2.76 mmol/L, IQR: 2.25 to 3.20 mmol/L vs median: 2.52 mmol/L, IQR: 2.02 to 2.87 mmol/L, P5.001, respectively) in patients with CHD.
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Figure 1
Table 2. Correlations Within the Serum Levels of Various Analytes and Lipid Profiles in Patients With CHDa Analyte hs-CRP Spearman correlation P value GFR Spearman correlation P value Cys C Spearman correlation P value
GFR
hs-CRP
BUN
SCr
UA
TC
TG
HDL-c
LDL-c
NA NA
NA NA
0.091 .04
0.034 .44
0.008 .86
0.018 .68
0.138 .002
0.137 .002
0.151 .001
NA NA
0.057 .19
0.305 5.001
0.850 5.001
0.321 5.001
0.013 .77
0.106 .02
0.071 .10
0.132 .002
0.075 .09
0.420 5.001
0.017 .70
0.076 .08
0.039 .37
0.017 .70
0.103 .02
0.132 .002
0.066 .13
CHD, coronary heart disease; GFR, glomerular filtration rate; hs-CRP, high-sensitivity C-reactive protein; BUN, blood urine nitrogen; SCr, serum creatinine; UA, uric acid; TC, total cholesterol; TG, triglyceride; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; Cys C, cystatin C. a Bolding indicates statistical significance.
Logistic Regression Analysis of Independent Predictors of CHD Prevalence To assess the factors predicting the prevalent risk of CHD, we performed univariate and multivariate logistic regression analyses. Results of the univariate analysis showed that serum levels of TC, TG, HDL-c, LDL-c, hs-CRP, and cystatin C were associated with the prevalence of CHD (Table 3). Further, the results of multivariate analysis indicated that the level of cystatin C (OR ¼ 1.648, 95% confidence interval [CI], 1.454 to 1.868, P5.001; Table 3) was the strongest independent predictor of CHD risk in the studied cohort.
Discussion CKD has been recognized as a significant burden for patients with CHD. In patients with CHD, comorbidities of
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CKD further increased the risk of incidence of major adverse cardiovascular events and death. Currently, the diagnosis of CKD is based on GFR values, which are calculated primarily from serum concentrations of SCr. However, SCr has been found to be affected by many factors other than renal function, such as age, sex, muscle mass, and physicalactivity levels.6,10-12 In fact, patients with a mild decline in renal function, GFR evaluation based on SCr level might be biased. A study23 indicated that cystatin C, a protein that belongs to the family of cysteine proteinase inhibitors and is synthesized in a stable manner by almost all nucleated cells, may better reflect renal function.23 Further, cystatin C is a highly sensitive marker for renal function, reflecting preclinical renal dysfunction when SCr levels and GFR remain normal. Elevated serum cystatin C levels are related to increased risk of CHD24 and severity of coronary atherosclerotic lesions,25,26 particularly in patients with CKD. However, the association of cystatin C and CHD risk in patients without CKD has not been fully investigated. 27
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Serum levels of analytes in control subjects and patients with coronary heart disease (CHD). A, Cystatin C levels. B, High-sensitivity C-reactive protein (hs-CRP). The values were presented as medians and the interquartile ranges because these data were not normally distributed.
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Scatter diagrams of comparative data from our cohort. These diagrams illustrate the correlation between glomular filtration rate (GFR) and levels of blood urea nitrogen (BUN) (A), serum creatinine (SCr) (B), uric acid (UA) (C), low-density lipoprotein cholesterol (LDL-c) (D), and serum cystatin C (E); and between levels of serum cystatin C and high-sensitivity C-reactive protein (hs-CRP) (F).
Table 3. Univariate and Multivariate Logistic Regression Analysis of Factors Predicting CHD Prevalencea Variable
Age Sex TC TG HDL-c LDL-c ALT AST T-bil BUN SCr UA GFR(mlmin1 1.73 m2) Cys C hs-CRP
Univariate
Multivariate
OR (95% CI)
P Value
OR (95% CI)
P Value
1.002 (.980-1.024) 1.227 (.787-1.913) 1.328 (1.078-1.635) 1.922 (1.466-2.521) 0.170 (.078-.370) 1.993 (1.503-2.643) 1.008 (0.996-1.021) 1.013 (0.999-1.027) 1.028 (0.990-1.067) 0.957 (0.835-1.096) 0.946 (0.373-2.396) 1.001 (0.999-1.004) 1.000 (0.992-1.007) 1.703 (1.508-1.922) 1.154 (1.087-1.226)
.85 .37 .008 5.001 5.001 5.001 .21 .06 .15 .53 .91 .29 .94 5.001 5.001
NA NA 0.888 (0.634-1.245) 1.472 (1.065-2.035) 0.371 (0.136-1.013) 1.749 (1.109-2.758) NA NA NA NA NA NA NA 1.648 (1.454-1.868) 1.031 (0.968-1.097)
NA NA .49 .02 .05 .02 NA NA NA NA NA NA NA 5.001 .34
OR, odds ratio; CI, confidence interval; CHD, coronary heart disease; TC, total cholesterol; TG, triglyceride; HDL-c, high-density lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; ALT, alanine aminotransferase; AST, aspartate aminotransferase; T-bil, total bilirubin; BUN, blood urine nitrogen; SCr, serum creatinine; UA, uric acid; GFR, glomerular filtration rate; Cys C, cystatin C; hs-CRP, high-sensitivity C-reactive protein. a Bolding indicates statistical significance.
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Figure 2
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Despite these findings, it is currently unclear whether cystatin C is a direct marker of cardiovascular diseases or only a marker of preclinical renal dysfunction in our cohort. Inflammation plays an important role in the pathogenesis and prognosis of CHD. The results of our study and other studies29,30 have suggested a close correlation between cystatin C and the conventional inflammatory maker hs-CRP. Therefore, it is reasonable to suggest that the cystatin C level may be of predictive value in CHD by reflecting the amount of systemic inflammation. Indeed, examination of the relationship between serum cystatin C and frequent inflammatory response in patients with declined renal function has been reported in the literature.31 However, a possible explanation for our results is that serum cystatin C is a more sensitive marker of early renal dysfunction, which may be involved in the process of CHD. Despite the uncertainty of the exact mechanisms underlying the predictive role of cystatin C in CHD, evidence suggests that elevated serum cystatin C is associated with worse prognosis in patients with CHD. A study by Zethelius et al32 assessed whether a combination of biomarkers, including cystatin C, N-terminal pro-brain natriuretic peptide, troponin, and hs-CRP, improved patient risk stratification compared with established cardiovascular risk factors. Those researchers discovered that adding cystatin C to the system significantly improved predictive efficacy.32 More direct evidence may be derived from animal studies or pathologic studies in humans, which have demonstrated that the cystatin C levels were decreased in tissues with atherosclerotic plaques and aneurysms.33,34 Also, a recent study report by Niccoli et al35 revealed that the concentration of blood cystatin C was directly proportional to the number of
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stenotic lesions in 70 patients with acute coronary syndrome and that cystatin C was associated with severity of coronary atherosclerosis plaque formation. Further studies are needed to confirm the precise mechanisms underlying the predictive and therapeutic value of cystatin C in CHD. Moreover, cystatin C seems to offer more complete prognostic information than other markers of renal disease and is one of the strongest risk predictors of CHD. Plasma cystatin C is a marker for CKD, a disease strongly associated with an increased risk for cardiovascular disease.36,37 Because of the association of renal dysfunction with cardiovascular disease, it is unclear whether cystatin C is a direct marker of CHD or merely a marker for renal dysfunction, which has implications for therapeutic intervention. Cystatin C is measured via particle-enhanced immunonephelometric assay, using particles coated with cystatin C–specific antibodies, and subsequent turbidimetry or nephelometry.38 The assays are precise: detection methods provide coefficients of variation ranging from 2% to 8%.39 Otherwise, the intra- and interindividual biologic variations of serum cystatin C are 5% and 13%, respectively.40 Our data showed that the serum cystatin C concentrations of patients with CHD were elevated by more than 20% compared with controls. These data could be used clinically and could imply clinical significance. The results of our study indicate that elevated serum cystatin C level is significantly associated with the risk prevalence for CHD in patients without CKD, suggesting that even subtle renal dysfunction, which cannot be detected via SCr levels, is associated with the incidence of CHD. Because the presence of CHD is one of the major determinants of poor prognosis in patients with impaired renal function, serum levels of cystatin C are expected to be useful in identifying patients at risk for CHD, even in the absence of CKD. LM
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