Cystatin-C and inflammatory markers in the ambulatory elderly

Transcription

1 The American Journal of Medicine (2005) 118, 1416.e e31 CLINICAL RESEARCH STUDY Cystatin-C and inflammatory markers in the ambulatory elderly Michael G. Shlipak, MD, MPH, a Ronit Katz, PhD, b Mary Cushman, MD, c Mark J. Sarnak, MD, MS, d Catherine Stehman-Breen, MD, MS, e Bruce M. Psaty, MD, MPH, f David Siscovick, MD, MPH, f Russell P. Tracy, PhD, c Anne Newman, MD, MPH, g Linda Fried, MD, MPH h a General Internal Medicine Section, San Francisco Veterans Affairs Medical Center and Departments of Medicine, Epidemiology and Biostatistics, University of California, San Francisco, Calif; b Collaborative Health Studies Coordinating Center, Seattle Wash; c Departments of Medicine and Pathology, University of Vermont, Burlington, Vt; d Division of Nephrology, Department of Medicine, Tufts-New England Medical Center, Boston, Mass; e Amgen Inc., Thousand Oaks, Calif; f Departments of Medicine and Epidemiology, University of Washington, Seattle, Wash; g Department of Epidemiology, University of Pittsburgh Graduate School of Public Health, and Division of Geriatric Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa; and h Renal Section, Medical Service, VA Pittsburgh Healthcare System, Pittsburgh, Pa. KEYWORDS: Kidney disease; Cystatin-C; Inflammation; C-reactive protein; Fibrinogen; Creatinine ABSTRACT PURPOSE: Inflammatory factors are elevated in persons with severe renal dysfunction, but their association across all levels of renal function is unclear. We compared cystatin-c, a novel marker of renal function, with creatinine and estimated glomerular filtration rate (egfr) as predictors of C-reactive protein and fibrinogen levels. METHODS: This study is a cross-sectional analysis to evaluate cystatin-c, creatinine, and egfr as predictors of the inflammatory markers C-reactive protein and fibrinogen. Participants included 4637 ambulatory elderly patients from the Cardiovascular Health Study. Multivariate linear regression was used to determine the independent associations of each renal function measurement with the inflammatory marker outcomes. RESULTS: After adjustment for confounding factors, cystatin-c was correlated with both C-reactive protein (coefficient 0.13; 95% confidence interval: , P.0001) and fibrinogen levels (0.15; , P.0001). Associations were larger than those for creatinine and C-reactive protein (0.05; , P.003) or fibrinogen (0.07; , P.0001). Adjusted levels of C-reactive protein increased incrementally across quintiles of cystatin-c, from a median of 2.2 mg/l in quintile 1 to 3.7 mg/l in quintile 5. In contrast, both C-reactive protein and fibrinogen had U-shaped associations with quintiles of creatinine and egfr, because the inflammatory markers were equivalently elevated in quintiles 1 and 5. CONCLUSIONS: The finding of a significant linear association of cystatin-c and inflammation markers suggests that even small reductions in renal function may be associated with adverse pathophysiologic consequences Elsevier Inc. All rights reserved /$ -see front matter 2005 Elsevier Inc. All rights reserved. doi: /j.amjmed

2 1416.e26 The American Journal of Medicine, Vol 118, No 12, December 2005 Chronic kidney disease is an independent predictor of cardiovascular morbidity and mortality, but the mechanisms underlying this association remain unclear. One potential mediator for cardiovascular risk in the setting of chronic kidney disease is the presence of increased inflammation. A prior study from the Cardiovascular Health Study (CHS) reported that levels of inflammatory and procoagulant biomarkers were moderately elevated among elderly participants with creatinine clearance less than 40 ml/min (Cockroft-Gault equation), but there was no difference in levels between those with creatinine clearance 40 to 60 ml/min and those with creatinine clearance greater than 60 ml/ min. 1 An analysis from the National Health and Nutrition Examination Survey III similarly found that participants with an estimated glomerular filtration rate (egfr) less than 60 ml min 1.73 m 2 (Modification of Diet in Renal Disease equation) had elevated levels of C-reactive protein and fibrinogen compared with participants with an egfr 60 to 89 ml min 1.73 m 2 or greater than 90 ml min 1.73 m 2. 2 The results of these studies suggest a threshold effect whereby inflammatory biomarkers may accumulate only in persons with moderate to severe renal dysfunction. An alternative possibility is that creatinine-based estimates of GFR are not capable of distinguishing gradients in renal function within the presumed normal range. 3 Thus, an association of inflammatory markers with moderately impaired levels of renal function may have been missed. Cystatin-C is a novel marker of renal function that has a more linear association with GFR than creatinine or creatininebased estimates of GFR. 4-6 Cystatin-C has been shown to have stronger and more linear associations with heart failure, mortality, and cardiovascular risk compared with creatinine and egfr. 7-9 On the basis of those findings, we hypothesized that levels of inflammatory markers might also be linearly associated with renal function. In this study, we evaluated the associations of cystatin-c, creatinine, and egfr with C-reactive protein and fibrinogen concentrations among participants in CHS, a cohort of ambulatory elderly persons. Materials and methods Design This is a cross-sectional study conducted from the 1992 to 1993 visit of the CHS. The CHS is a community-based, longitudinal study of adults aged 65 years or more at entry with adherence to the Declaration of Helsinki. The objective Reprint requests should be addressed to Michael G. Shlipak, MD, MPH, General Internal Medicine Section, VA Medical Center (111A1), 4150 Clement St., San Francisco, CA Manuscript submitted April 13, 2005, and accepted in revised form July 28, address:shlip@itsa.ucsf.edu of the study was to evaluate risk factors for the development and progression of cardiovascular disease. 10 The original 5201 study participants were recruited between 1989 and 1990 from 4 US communities: Sacramento County, Calif; Forsyth County, NC; Washington County, Md; and Allegheny County, Pa. 11 An additional 687 African Americans were recruited in 1992 and Eligible participants included individuals sampled at random from Medicare eligibility lists in their respective area and their spouses. Of the total 5888 study participants enrolled in CHS, cystatin-c, creatinine, C-reactive protein, and fibrinogen measurements were available and analyzed in 4637 patients. Measurements Renal function was measured by serum cystatin-c and creatinine levels, and by creatinine-based egfr, using the simplified Modification of Diet in Renal Disease equation. 12 All assays were measured on serum drawn in the morning and stored at 70 C. Cystatin-C was measured in 2003 in a BNII nephelometer (Dade Behring Inc., Deerfield, Ill) using a particle-enhanced immunonepholometric assay (N Latex Cystatin-C). 13 Polystyrene particles were coated with monoclonal antibodies to cystatin-c that agglutinate in the presence of antigen (cystatin-c) to cause an increase in the intensity of scattered light in proportion to the amount of cystatin-c in the sample. The assay range is to mg/l, with the reference range for young, healthy individuals reported as 0.53 to 0.95 mg/l. Intra-assay coefficients of variation range from 2.0% to 2.8%, and interassay coefficients of variation range from 2.3% to 3.1%. Serum creatinine levels were measured at the time of the 1992 to 1993 annual visit using the Kodak Ektachem 700 Analyzer (Eastman Kodak, Rochester, NY), a colorimetric method. Inflammatory markers The outcomes of this study were the serum levels of C-reactive protein and fibrinogen, which were measured using samples from the 1992 to 1993 visit. C-reactive protein was measured on plasma using an automated assay on the BNII nephelometer (Dade Behring Inc.) 14 Fibrinogen was measured in a BBL fibrometer (Becton Dickinson, Cockeysville, Md). 15,16 Covariates considered for statistical adjustment included age, sex, race, smoking status (current, former/never), body mass index, alcohol use (drinks/week), history of hypertension, coronary heart disease (prior myocardial infarction, angina, angioplasty, or bypass surgery), heart failure, left ventricular hypertrophy (by electrocardiogram), and stroke; and levels of low-density lipoprotein cholesterol (calculated), high-density lipoprotein cholesterol, and glucose. Analysis We initially performed univariate correlations of each measure of renal function with the inflammatory markers. Sep-

3 Shlipak et al Cystatin-C and inflammation 1416.e27 Table 1 Unadjusted and multivariate adjusted linear correlations of renal function measurements with C-reactive protein and fibrinogen levels Cystatin-C Serum creatinine egfr 95% CI P value 95% CI P value 95% CI P value Log(CRP) Unadjusted (0.166, 0.220) (0.039, 0.097) ( 0.035, 0.021).650 Adjusted* (0.128, 0.188) (0.024, 0.085) ( 0.049, 0.014).278 Adjusted (0.099, 0.155) (0.017, 0.073) ( 0.048, 0.008).191 Fibrinogen Unadjusted (0.168, 0.222) (0.064, 0.120) ( 0.077, 0.021).001 Adjusted* (0.146, 0.210) (0.048, 0.118) ( 0.067, 0.002).037 Adjusted (0.126, 0.181) (0.042, 0.098) ( 0.059, 0.003).045 egfr estimated glomerular filtration rate; CRP C-reactive protein; BMI body mass index; ECG LVH left ventricular hypertrophy by electrocardiography; LDL low-density lipoprotein; HDL high-density lipoprotein. *Adjusted for age, gender, race, and BMI. Adjusted using multivariate linear regression for age, gender, race, BMI, hypertension, smoking status, alcohol intake, ECG LVH, LDL, HDL, glucose, stroke, heart failure, and coronary heart disease. arately, for each measure of renal function, we conducted multivariate linear regressions that adjusted for all of the covariates listed above. In these models, we log-transformed C-reactive protein levels because their distribution was skewed to the right. The results of these models were also used to compare the strength of association of the various correlates of inflammation, including cystatin-c. To compare levels of each inflammatory marker across the distribution of each renal function measurement, we categorized each renal measure into quintiles and determined the median level of C-reactive protein and mean level of fibrinogen within each quintile. The adjusted mean levels were calculated from the linear regression Y adj.mean Y mean b(x ith.mean X mean ), where Y is the interval dependent, Figure 1 Estimated median level of C-reactive protein (CRP) and mean level of fibrinogen by cystatin-c quintile. Cutpoints for quintiles of cystatin-c: 0.90, , , , 1.28 mg/l.

4 1416.e28 The American Journal of Medicine, Vol 118, No 12, December 2005 Figure 2 Estimated median level of C-reactive protein (CRP) and mean level of fibrinogen by creatinine quintile. Cutpoints for quintiles of creatinine: (in men) 0.86, , , , 1.25 mg/dl; (in women) 0.66, , , , 0.95 mg/dl. X is the covariate, i is one of the k groups, and b is the regression coefficient. For each covariate, there is an additional X term in the equation. To obtain the median adjusted levels of C-reactive protein, we calculated the adjusted mean level of log C-reactive protein (using the above method) and then took the anti-log of our estimates. To compare the strength of correlation with each biomarker among the multiple covariates, we used standardized coefficients for linear variables. These are regression coefficients from a multivariate model that are fitted to standardized data; each observation was subtracted from the cohort mean for that measurement (eg, high-density lipoprotein cholesterol) and divided by the cohort standard deviation. Results Serum levels of cystatin-c, creatinine, C-reactive protein, and fibrinogen were measured in 4637 participants at the 1992 to 1993 visit of the CHS. Among these participants, the mean age was 75 years; 58% were women; and the racial distribution was white (82%), African American (17%), and other (1%). In unadjusted analyses, cystatin-c had significant correlations with both log(crp) and fibrinogen levels (Table 1). These associations were somewhat attenuated in multivariate linear regression analyses. Serum creatinine levels were also associated with both log(crp) and fibrinogen levels in unadjusted and adjusted analyses, but the point estimates were much closer to the null than those of cystatin-c. The 95% confidence intervals comparing the associations of cystatin-c and creatinine with each inflammatory marker did not overlap. Estimated GFR was not significantly associated with log(crp) levels, but was weakly associated with fibrinogen. In an analysis of median C-reactive protein and mean fibrinogen levels across quintiles of each renal measure, both C-reactive protein and fibrinogen levels were incrementally higher across quintiles of cystatin-c. The unadjusted median C-reactive protein concentration ranged from 2.0 mg/l in the lowest quintile to 4.0 mg/l in the highest; multivariate adjustment only minimally diminished this association, with adjusted median levels of 2.2 mg/l in the lowest quintile and 3.7 mg/l in the highest quintile (Figure 1). Similarly, the adjusted mean fibrinogen concentration was higher with each quintile of cystatin-c, from 313 mg/dl in the lowest quintile to 356 mg/dl in the highest quintile (Figure 1). In contrast, the association of both creatinine and egfr quintiles with both inflammatory markers was U-shaped with little difference between the

5 Shlipak et al Cystatin-C and inflammation 1416.e29 Figure 3 Estimated median level of C-reactive protein (CRP) and mean level of fibrinogen by estimated glomerular filtration rate (egfr) quintile. Cutpoints for egfr: 82.8, , , , 55.7 ml min 1.73 m 2. low and high quintiles. For creatinine, the lowest adjusted levels of C-reactive protein (2.6 mg/l) were observed in the second and third quintiles and the lowest fibrinogen levels (325 mg/dl) were in the second quintile; the fifth quintile had the highest median C-reactive protein (3.0 mg/l) and mean fibrinogen (341 mg/dl) levels (Figure 2). Among egfr quintiles, C-reactive protein levels were highest in the first and fifth quintiles (3.0 mg/l and 2.9 mg/l, respectively), and equally low in the third and fourth quintiles (2.6 mg/l) (Figure 3). We compared the associations of all the correlates of log(crp) and fibrinogen levels using multivariate linear regression to determine the relative strength of cystatin-c. On the basis of the adjusted, standardized coefficients, cystatin-c was the second strongest correlate of log(crp) after body mass index, and cystatin-c was the strongest correlate of fibrinogen (Table 2). Discussion In this study of ambulatory elderly adults, cystatin-c, a novel measure of renal function, had a significant and linear association with levels of the inflammatory biomarkers C- reactive protein and fibrinogen. Among all predictors evaluated, cystatin-c was the strongest correlate of fibrinogen and the second strongest correlate of C-reactive protein. In contrast, creatinine and creatinine-based egfr had a much weaker and nonlinear association with the inflammation biomarkers. These findings challenge prior studies suggesting that inflammatory factors were only associated with kidney function below some threshold of GFR. 1,2 The association of end-stage renal disease and severe pre-dialysis renal insufficiency with inflammation has been established in several studies Recently, 2 studies found mild-to-moderate renal insufficiency to be associated with elevated levels of inflammatory biomarkers, but only among subjects with an egfr less than 60 ml min 1.73 m 2. 1,2 Because both studies used creatininebased estimates of GFR to determine renal function, they probably could not distinguish renal function impairment among subjects with presumed normal GFR. Because cystatin-c is not a breakdown product of muscle and does not vary with age, sex, and lean muscle mass, we were able to identify a much more graded association of renal function with C-reactive protein and fibrinogen than was possible using creatinine or egfr. Presumably, the association of cystatin-c with these inflammatory biomarkers reflects the underlying association of cystatin-c with kidney function. Several studies have

6 1416.e30 The American Journal of Medicine, Vol 118, No 12, December 2005 Table 2 Log(CRP) Independent predictors of C-reactive protein and fibrinogen levels, ranked by strength of association Fibrinogen Covariate Standardized coefficient* P value Covariate Standardized coefficient* P value BMI (kg/m 2 ) Cystatin-C (mg/l) Cystatin-C (mg/l) LDL (mg/dl) Male sex Non-white race Current smoking Smoking status AAI AAI HDL (mg/dl) HDL (mg/dl) Age BMI (kg/m 2 ) Glucose (mg/dl) Heart failure Heart failure Male sex Coronary heart disease Alcohol use (drinks/wk) Non-white race Coronary heart disease Left ventricular hypertrophy Hypertension Prior stroke Alcohol use (drinks/wk) CRP C-reactive protein; BMI body mass index; AAI ankle arm index; HDL high-density lipoprotein; LDL low-density lipoprotein. *Standardized coefficients are regression coefficients from a multivariate model fitted to standardized data; each observation was subtracted from the cohort mean for that measurement (eg, HDL) and divided by the cohort standard deviation. reported that cystatin-c has a stronger association with measured GFR than serum creatinine 4 or creatinine estimates of GFR. 5,6 Furthermore, in a 4-year longitudinal study in persons with diabetes, but without chronic kidney disease, Perkins and colleagues 21 found that cystatin-c correlated tightly with serial measurements of GFR by iothalamate clearance, whereas the creatinine-based Modification of Diet in Renal Disease was a much weaker reflection of GFR. We have no evidence to suggest that cystatin-c is itself a marker of inflammation, other then being a proxy of kidney function. A recent editorial, 22 however, suggested that cystatin-c s association with cardiovascular outcomes was caused by its role as an inflammatory marker, although those analyses were adjusted for C-reactive protein, fibrinogen, albumin, and factor VIII concentration. 8,9,23 Knight and colleagues 24 have also reported that cystatin-c was influenced by several nonrenal factors, but they compared cystatin-c with creatinine clearance rather than the gold standard of directly measured GFR. Creatinine clearance is known to be an imprecise measure of GFR. 25 This study has limitations that should be considered. Most importantly, it was cross-sectional, so we cannot determine the direction of the association between cystatin-c and inflammation. In addition, we may have missed underlying confounders that would explain the association of cystatin-c with inflammation; conversely, we may have also underestimated the effect of kidney function by adjusting for characteristics, such as hypertension, that may be a consequence of kidney disease. Cystatin-C and creatinine assays were not performed at the same time, although cystatin-c levels seem stable after several years of frozen storage. 26 The substantial differences between the associations of cystatin-c and creatinine with inflammatory markers observed in CHS may be smaller in other populations, such as the non-elderly. In summary, cystatin-c levels are a significant linear correlate of the inflammatory biomarkers C-reactive protein and fibrinogen. These findings suggest that even small reductions in renal function may be associated with adverse physiology. In research settings cystatin-c may offer the opportunity to evaluate the physiologic correlates of renal function across a broad spectrum that would not have been distinguishable with creatinine. Future studies should address whether cystatin-c will have a role in clinical medicine. Acknowledgments Drs. Shlipak, Fried, and Katz are funded by R01 HL Dr. Shlipak is also supported by the American Federation for Aging Research and National Institute on Aging (Paul Beeson Scholars Program) and the Robert Wood Johnson Foundation (Generalist Faculty Scholars Program). Dr. Sarnak is supported by a K23 award from the National Institute of Diabetes and Digestive and Kidney Diseases. The CHS Study is supported by contracts N01-HC to N01-HC-85086, N01-HC-35129, and N01 HC from the National Heart, Lung, and Blood Institute. A full list of participating CHS investigators and institutions can be found at

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