CLINICAL RESEARCH www.jasn.org

HDL , , and Cardiovascular Risk in Hemodialysis Patients

†‡ | Günther Silbernagel,* Bernd Genser, § Christiane Drechsler,§ Hubert Scharnagl,¶ † | | Tanja B. Grammer, Tatjana Stojakovic,¶ Vera Krane,§ Eberhard Ritz,** Christoph Wanner,§ ††‡‡ and Winfried März¶

*Department of Angiology, Swiss Cardiovascular Center, Inselspital, University of Bern, Bern, Switzerland; †Mannheim Institute of Public Health, Social and Preventive Medicine, and ††Medical Clinic V (Nephrology, Hypertensiology, Endocrinology, Diabetology, Rheumatology), Department of Internal Medicine, Mannheim Medical Faculty, University of Heidelberg, Mannheim, Germany; ‡Institute of Public Health, Federal University of Bahia, Salvador, Brazil; §Division of Nephrology, Department of Medicine I, and |Comprehensive Heart Failure Centre, University of Würzburg, Würzburg, Germany; ¶Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; **Division of Nephrology, Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany; and ‡‡Synlab Academy, Synlab Services LLC, Mannheim, Germany

ABSTRACT High concentrations of HDL cholesterol are considered to indicate efficient reverse cholesterol transport and to protect from atherosclerosis. However, HDL has been suggested to be dysfunctional in ESRD. Hence, our main objective was to investigate the effect of HDL cholesterol on outcomes in maintenance hemodialysis patients with diabetes. Moreover, we investigated the associations between the major components of HDL (apoA1, apoA2, and apoC3) and end points. We performed an exploratory, post hoc analysis with 1255 participants (677 men and 578 women) of the German Diabetes Dialysis study. The mean age was 66.3 years and the mean body mass index was 28.0 kg/m2.Theprimary end point was a composite of cardiac death, myocardial infarction, and stroke. The secondary end point included all-cause mortality. The mean duration of follow-up was 3.9 years. A total of 31.3% of the study participants reached the primary end point and 49.1% died from any cause. HDL cholesterol and apoA1 and apoC3 quartiles were not related to end points. However, there was a trend toward an inverse association between apoA2 and all-cause mortality. The hazard ratio for death from any cause in the fourth quartile compared with the first quartile of apoA2 was 0.63 (95% confidence interval, 0.40 to 0.89). The lack of an association between HDL cholesterol and cardiovascular risk may support the concept of dysfunctional HDL in hemodialysis. The possible beneficial effect of apoA2 on survival requires confir- mation in future studies.

J Am Soc Nephrol 26: 484–492, 2015. doi: 10.1681/ASN.2013080816

Reverse cholesterol transport is considered to pro- Received August 1, 2013. Accepted June 1, 2014. 1,2 tect from atherosclerosis. HDLs represent the G.S. and B.G. contributed equally to this work. major vehicle of reverse cholesterol transport.1,2 They primarily consist of phospholipids, choles- Published online ahead of print. Publication date available at www.jasn.org. terol, and .1,2 The two major protein con- stituents of HDL are apoA1 and apoA2.1,2 ApoC3 Correspondence: Dr. Günther Silbernagel, Department of Angiology, Swiss Cardiovascular Center, Inselspital, University 3,4 forms another component of HDL. Whereas of Bern, 3010 Bern, Switzerland. Email: guenther.silbernagel@ apoA1 is regarded as beneficial to cardiovascular insel.ch health, apoC3 is rather regarded as proathero- – genic.3 6 The role of apoA2 has not been sufficiently Copyright © 2015 by the American Society of Nephrology

484 ISSN : 1046-6673/2602-484 J Am Soc Nephrol 26: 484–492, 2015 www.jasn.org CLINICAL RESEARCH examined.7,8 Nevertheless, recent data tend to support anti- RESULTS atherogenic effects.7–9 High concentrations of HDL cholesterol are generally Patient Characteristics at Baseline thought to reflect efficient reverse cholesterol transport.2,5 The 1255 (677 men and 578 women) participants of the 4D However, HDL composition and function are impaired in study had a mean age of 66.3 years and a mean body mass index CKD.10,11 In addition, dysfunctional HDL has been observed of 28.0 kg/m2. in patients on hemodialysis.12 Hence, high HDL cholesterol Tables 1–4 show the baseline characteristics of the study does not necessarily indicate efficient reverse cholesterol participants according to the quartiles of HDL cholesterol and transport in renal disease. apoA1, apoA2, and apoC3. Supplemental Table 1 shows the We conducted an exploratory, post hoc analysis of the baseline characteristics of the subgroups with HDL cholesterol German Diabetes Dialysis (4D) study to investigate whether levels #50 mg/dl and .50 mg/dl within the fourth quartile of HDL cholesterol predicts mortality and cardiovascular risk in HDL cholesterol. Supplemental Table 2 shows the baseline patients on maintenance hemodialysis treatment.13,14 More- characteristics of the propensity score–matched cohort. over, we sought to analyze the relationships of the major pro- In the entire cohort, the HDL cholesterol quartiles were tein components of HDL, namely apoA1, apoA2, and apoC3, inversely related to the proportion of men, to body mass index, with outcomes. , apoC3, and C-reactive protein and they were

Table 1. Baseline patient characteristics according to quartiles of HDL cholesterol Quartile of HDL Cholesterol Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 312 308 317 318 Range (mg/dl) 17.3–33.4 33.5–39.3 39.4–46.9 47.0–115.5 Age (yr) 65.3 (8.3) 66.0 (8.6) 67.2 (8.1) 66.7 (8.0) 0.03 Men (%) 205 (66) 185 (60) 156 (49) 131 (41) ,0.001 Ever smoking, n (%) 146 (47) 123 (40) 120 (38) 118 (37) 0.06 Body mass index (kg/m2) 30.0 (4.7) 28.2 (5.0) 27.7 (5.0) 26.2 (4.3) ,0.001 BP (mmHg) Systolic 145 (22) 147 (22) 144 (21) 147 (22) 0.19 Diastolic 75 (11) 76 (12) 76 (11) 77 (11) 0.13 Time receiving dialysis (mo) 8.9 (6.7) 8.3 (7.2) 8.1 (7.0) 7.8 (6.6) 0.24 History of disease, n (%)a Arrhythmia 76 (24) 52 (17) 49 (15) 59 (19) 0.03 MI, CABG, PCI, or CHD 103 (33) 94 (31) 79 (25) 93 (29) 0.16 Congestive heart failureb 132 (42) 107 (35) 102 (32) 103 (32) 0.03 Stroke or TIA 57 (18) 63 (20) 55 (17) 49 (15) 0.42 Peripheral vascular disease 148 (47) 147 (48) 125 (39) 140 (44) 0.13 Hemoglobin (g/dl) 10.8 (1.4) 11.0 (1.3) 10.8 (1.3) 11.0 (1.4) 0.08 Glycated hemoglobin (%) 6.84 (1.31) 6.72 (1.24) 6.61 (1.27) 6.72 (1.21) 0.15 Phosphate (mg/dl) 5.98 (1.72) 6.07 (1.56) 5.97 (1.51) 6.09 (1.63) 0.71 Albumin (g/dl) 3.83 (0.3) 3.81 (0.26) 3.80 (0.32) 3.81 (0.31) 0.47 Cholesterol (mg/dl) LDL 119.4 (26.9) 125.0 (30.2) 128.3 (30.4) 129.5 (40.0) ,0.001 HDL 29.6 (3.0) 36.1 (1.7) 43.0 (2.2) 56.9 (9.6) ,0.001 (mg/dl) A1 107.9 (14.4) 118.5 (16.8) 129.0 (16.1) 149.5 (23.2) ,0.001 A2 24.5 (4.4) 27.1 (4.5) 29.1 (4.9) 32.2 (6.1) ,0.001 C3 22.3 (10.6) 20.8 (10.0) 19.5 (8.8) 19.1 (8.1) ,0.001 Triglycerides (mg/dl) 349.0 (200.7) 291.6 (170.1) 236.2 (134.1) 181.4 (95.6) ,0.001 C-reactive protein (mg/L) 11.0 (4.3–13.6) 6.6 (2.8–12.0) 5.7 (2.5–11.7) 4.5 (1.9–11.1) ,0.001 NT-pro-BNP (ng/L) 3585 (1492–9949) 3117 (1416–6842) 2698 (1246–8375) 3953 (1686–11,115) 0.03 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II.

J Am Soc Nephrol 26: 484–492, 2015 HDL Cholesterol and Outcomes in 4D 485 CLINICAL RESEARCH www.jasn.org positively related to LDL cholesterol and apoA1 and apoA2 different end points according to HDL cholesterol and apolipo- (Table 1). The apoA1 quartiles were inversely related to the protein quartiles are shown in Supplemental Tables 3–6. The proportion of men, triglycerides, and C-reactive protein and interaction terms between components of HDL and randomi- they were positively associated with albumin, LDL cholesterol, zation group were not significant for any end point in pooled and apopliproteins (Table 2). The apoA2 quartiles were inversely analyses. These findings were corroborated by analyses stratified related to C-reactive protein, N-terminal pro-brain natriuretic by randomization group (data not shown). peptide, congestive heart failure, and arrhythmia and they were Table 5 shows a summary of the pooled time-to-event anal- positively associated with hemoglobin, albumin, and LDL cho- yses. The quartiles of HDL cholesterol were not associated lesterol (Table 3). The apoC3 quartiles were inversely related to with any end point (Table 5). Further stratification of the age and N-terminal pro-brain natriuretic peptide and they were fourth quartile in patients with HDL cholesterol #50 mg/dl positively associated with body mass index, glycated hemoglo- and in those with HDL cholesterol .50 mg/dl did not reveal bin, LDL cholesterol, and triglycerides (Table 4). significant associations (Supplemental Table 7). Adjustment for HDL cholesterol measurements during the follow-up did Effect of Baseline HDL Cholesterol and Apolipoprotein not change the results (Supplemental Table 8). The cubic Concentrations on Outcomes spline models corroborated the absence of any significant ef- A total of 31.3% of the study participants reached the com- fect of HDL cholesterol on both cardiovascular risk and all- posite vascular end point and 49.1% died from any cause. The cause death (Figure 1).

Table 2. Baseline patient characteristics according to quartiles of apoA1 Quartile of ApoA1 at Baseline Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 312 311 301 327 Range (mg/dl) 74–109 110–123 124–139 140–271 Age (yr) 65.6 (8.6) 66.3 (7.9) 67.1 (7.7) 66.2 (8.6) 0.13 Men (%) 208 (67) 188 (60) 147 (48) 134 (41) ,0.001 Ever smoking, n (%) 145 (46) 130 (42) 115 (38) 117 (36) 0.03 Body mass index (kg/m2) 27.5 (4.9) 27.7 (4.7) 27.6 (4.6) 27.3 (5.1) 0.66 BP (mmHg) Systolic 146 (22) 145 (23) 146 (22) 145 (21) 0.80 Diastolic 75 (10) 76 (11) 77 (11) 76 (11) 0.31 Time receiving dialysis (mo) 8.5 (7.1) 8.2 (6.5) 8.4 (7.2) 8.1 (6.8) 0.88 History of disease, n (%)a Arrhythmia 73 (23) 63 (20) 45 (15) 55 (17) 0.03 MI, CABG, PCI, or CHD 110 (35) 88 (28) 81 (27) 90 (28) 0.07 Congestive heart failureb 126 (40) 120 (39) 104 (34) 94 (29) 0.01 Stroke or TIA 61 (20) 48 (15) 59 (19) 56 (17) 0.49 Peripheral vascular disease 156 (50) 140 (45) 136 (45) 128 (39) 0.05 Hemoglobin (g/dl) 10.8 (1.5) 10.9 (1.3) 10.9 (1.3) 11.0 (1.4) 0.13 Glycated hemoglobin (%) 6.8 (1.3) 6.7 (1.2) 6.6 (1.2) 6.9 (1.3) 0.06 Phosphate (mg/dl) 5.98 (1.70) 5.96 (1.60) 6.02 (1.53) 6.16 (1.60) 0.37 Albumin (g/dl) 3.76 (0.29) 3.81 (0.31) 3.86 (0.30) 3.84 (0.29) ,0.001 Cholesterol (mg/dl) LDL 117 (26) 124 (26) 129 (30) 132 (31) ,0.001 HDL 26 (6) 32 (7) 38 (9) 49 (15) ,0.001 Apolipoprotein (mg/dl) A1 99.8 (6.8) 116.5 (4.0) 130.6 (4.8) 157.1 (18.4) ,0.001 A2 23.3 (3.6) 26.7 (3.5) 29.4 (4.1) 33.4 (5.8) ,0.001 C3 18.6 (9.7) 20.3 (9.4) 20.9 (9.2) 21.8 (9.5) ,0.001 Triglycerides (mg/dl) 292 (190) 268 (155) 263 (159) 235 (158) ,0.001 C-reactive protein (mg/L) 10.0 (3.7–15.8) 6.8 (3.1–11.2) 6.1 (2.6–11.1) 4.6 (1.9–11.1) ,0.001 NT-pro-BNP (ng/L) 4051 (1641–11,625) 2926 (1460–8322) 3076 (1351–7553) 2975 (1288–8040) 0.02 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II.

486 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 484–492, 2015 www.jasn.org CLINICAL RESEARCH

Table 3. Baseline patient characteristics according to quartiles of apoA2 Quartile of ApoA2 at Baseline Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 232 314 338 367 Range (mg/dl) 12–23 24–26 28–30 31–54 Age (yr) 66.3 (7.8) 66.7 (8.4) 66.4 (8.0) 65.9 (8.6) 0.58 Men (%) 133 (57) 176 (56) 172 (51) 193 (53) 0.37 Ever smoking, n (%) 98 (42) 131 (42) 135 (40) 140 (38) 0.72 Body mass index (kg/m2) 27.4 (4.7) 27.7 (4.8) 27.6 (5.0) 27.4 (4.6) 0.74 BP (mmHg) Systolic 144 (23) 147 (22) 145 (21) 146 (22) 0.51 Diastolic 75 (11) 75 (11) 77 (11) 76 (11) 0.17 Time receiving dialysis (mo) 8.4 (7.1) 8.3 (6.5) 8.7 (7.6) 7.8 (6.4) 0.47 History of disease, n (%)a Arrhythmia 64 (28) 66 (21) 48 (14) 57 (16) ,0.001 MI, CABG, PCI, or CHD 80 (34) 99 (31) 90 (27) 100 (27) 0.13 Congestive heart failureb 113 (49) 125 (40) 102 (30) 102 (28) ,0.001 Stroke or TIA 42 (18) 62 (20) 53 (16) 67 (18) 0.59 Peripheral vascular disease 121 (52) 151 (48) 146 (43) 141 (38) 0.01 Hemoglobin (g/dl) 10.6 (1.4) 10.9 (1.4) 11.0 (1.3) 11.1 (1.3) ,0.001 Glycated hemoglobin (%) 6.8 (1.2) 6.8 (1.3) 6.7 (1.2) 6.7 (1.2) 0.77 Phosphate (mg/dl) 6.00 (1.83) 6.00 (1.57) 6.09 (1.51) 6.02 (1.60) 0.86 Albumin (g/dl) 3.73 (0.31) 3.81 (0.30) 3.83 (0.29) 3.86 (0.30) ,0.001 Cholesterol (mg/dl) LDL 117 (29) 126 (27) 126 (29) 130 (32) ,0.001 HDL 28 (9) 32 (10) 37 (12) 44 (15) ,0.001 Apolipoprotein (mg/dl) A1 105.8 (13.6) 115.9 (15.6) 127.9 (16.9) 146.8 (23.4) ,0.001 A2 20.8 (2.1) 25.2 (1.0) 28.7 (1.0) 35.2 (4.1) ,0.001 C3 17.6 (10.2) 19.8 (9.3) 20.8 (9.1) 22.4 (9.1) ,0.001 Triglycerides (mg/dl) 291 (209) 271 (162) 264 (157) 241 (147) 0.004 C-reactive protein (mg/L) 11.1 (5.8–18.9) 8.4 (3.5–12.9) 5.1 (2.1–11.1) 4.4 (1.9–11.1) ,0.001 NT-pro-BNP (ng/L) 5713 (2558–13,735) 3415 (1402–8652) 2510 (1118–6649) 2742 (1310–6473) ,0.001 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II.

The quartiles of apoA1 and apoC3 were not associated with variable either (hazard ratio, 0.91; 95% confidence interval, end points (Table 5). By contrast, the multivariate adjusted 0.82 to 1.01; P=0.09). analyses conducted in the entire cohort showed that partici- pants in the fourth versus first quartile of apoA2 had decreased the risk of death from any cause (Table 5). The cubic spline DISCUSSION model corroborated the inverse association between apoA2 and the risk of death from any cause (P,0.001) (Figure 2). The main finding of this study was that the baseline concen- In the propensity score–matched subgroup, the hazard ratios trations of HDL cholesterol were not predictive of all-cause for all-cause mortality comparing the fourth, third, and sec- mortality and cardiovascular end points. The baseline con- ond with the first apoA2 quartile were similar to those centrations of apoA1 and apoC3 were also not associated with obtained in the entire cohort (see Supplemental Table 9). Be- outcomes. However, there was a trend toward an inverse causeofthelowersamplesize,theP value comparing the association between baseline apoA2 concentrations and death fourth with the first apoA2 quartile did not reach statistical from any cause. significance after Bonferroni correction in the subgroup anal- LDL lowering was demonstrated to reduce cardiovascular ysis. The association of apoA2 with the risk of death from any risk and mortality in the Study of Heart and Renal Protection cause did not reach statistical significance when apoA2 was study and in a post hoc analysis of the 4D study.15,16 Yet included in the Cox model as a standardized continuous lowering does not seem to be as effective in preventing

J Am Soc Nephrol 26: 484–492, 2015 HDL Cholesterol and Outcomes in 4D 487 CLINICAL RESEARCH www.jasn.org

Table 4. Baseline patient characteristics according to quartiles of apoC3 Quartile of Baseline ApoC3 Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 321 306 307 317 Range (mg/dl) 2–13 14–18 19–26 26–49 Age (yr) 67.1 (7.8) 67.7 (7.9) 65.9 (8.4) 64.6 (8.6) ,0.001 Men (%) 195 (61) 169 (55) 154 (50) 159 (50) 0.02 Ever smoking, n (%) 130 (41) 132 (43) 112 (36) 133 (41) 0.38 Body mass index (kg/m2) 26.4 (4.5) 26.6 (4.3) 28.2 (4.7) 29.0 (5.1) ,0.001 BP (mmHg) Systolic 146 (23) 146 (21) 147 (22) 143 (22) 0.25 Diastolic 76 (11) 75 (10) 76 (11) 75 (12) 0.14 Time receiving dialysis (mo) 7.7 (7.1) 7.8 (6.2) 9.1 (7.2) 8.6 (6.8) 0.03 History of disease, n (%)a Arrhythmia 68 (21) 63 (21) 53 (17) 52 (16) 0.29 MI, CABG, PCI, or CHD 98 (31) 82 (27) 91 (30) 98 (31) 0.71 Congestive heart failureb 123 (38) 114 (37) 99 (32) 108 (34) 0.33 Stroke or TIA 45 (14) 68 (22) 54 (16) 57 (18) 0.07 Peripheral vascular disease 146 (45) 145 (47) 139 (45) 130 (41) 0.35 Hemoglobin (g/dl) 10.8 (1.3) 11.0 (1.4) 11.0 (1.4) 10.8 (1.3) 0.12 Glycated hemoglobin (%) 6.6 (1.2) 6.5 (1.2) 6.8 (1.1) 7.0 (1.4) ,0.001 Phosphate (mg/dl) 5.75 (1.51) 6.04 (1.53) 6.19 (1.74) 6.15 (1.63) 0.002 Albumin (g/dl) 3.76 (0.32) 3.83 (0.27) 3.84 (0.30) 3.85 (0.31) 0.002 Cholesterol (mg/dl) LDL 114 (26) 126 (28) 133 (29) 130 (33) ,0.001 HDL 38 (13) 39 (12) 36 (14) 32 (133) ,0.001 Apolipoprotein (mg/dl) A1 120.5 (23.1) 127.7 (21.6) 127.6 (23.1) 129.6 (25.7) ,0.001 A2 26.1 (5.5) 28.8 (5.2) 28.9 (5.5) 29.3 (6.2) ,0.001 C3 10.1 (2.3) 15.9 (1.4) 21.9 (2.2) 33.9 (5.6) ,0.001 Triglycerides (mg/dl) 146.0 (61.0) 201.7 (77.7) 265.2 (102.1) 440.1 (202.1) ,0.001 C-reactive protein (mg/l) 6.8 (2.7–11.8) 5.0 (2.4–11.1) 7.1 (3.0–11.1) 7.4 (2.9–11.9) 0.02 NT-pro-BNP (ng/l) 5325 (1974–138,794) 3187 (1451–9503) 2659 (1171–7670) 2629 (1289–5207) ,0.001 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II. cardiovascular events in hemodialysis patients as in the general disease in another cohort of 607 hemodialysis patients.24 In population.14,17 However, patients on maintenance hemo- summary, the association of HDL cholesterol with outcomes dialysis treatment carry a very high cardiovascular risk.18 is at least weakened in ESRD. The data may therefore sup- Raising low levels of HDL cholesterol may reduce this risk.19 port the idea that HDL become dysfunctional in chronic The aforementioned hypothesis is also based on the consis- hemodialysis.25 tent relationship between low HDL cholesterol and increased Surprisingly, high apoA2, unlike apoA1, tended to be cardiovascular risk in the general population.5,20,21 However, associated with a lower risk of death from any cause in the this association did not hold true in these analyses. Few stud- 4D study. This observation may reflect differences in the ies have previously investigated the association between HDL metabolism of apoA1 and apoA2 in ESRD.26 Moreover, the as- cholesterol and apolipoproteins with outcomes in patients sociations of apoA2 with death from any cause may suggest with ESRD.22–24 Consistent with our results, HDL choles- potentially protective effects of this protein.7–9 In contrast terol was not associated with survival in a cohort of 15,859 with the lack of an association between apoA1 and end points hemodialysis patients from the DaVita dialysis clinics in the in our study, a previous study showed that induction of apoA1 United States.22 Oxidized HDL cholesterol was even demon- may improve HDL functionality in hemodialysis patients.27 strated to positively correlate with future cardiovascular Hence, future studies are needed to further elucidate the rela- events in 412 hemodialysis patients.23 By contrast, HDL cho- tionships of apoA1 and apoA2 with cardiovascular end points lesterol was inversely related to prevalent cardiovascular in ESRD.

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Table 5. Prognostic value of baseline lipid concentrations on risk of end point occurrence obtained by recurrent events time- to-event analysis HDL Cholesterol ApoA1 ApoA2 ApoC3 End point P P P P HR (95% CI)a HR (95% CI)a HR (95% CI)a HR (95% CI)a Valueb Valueb Valueb Valueb Combined vascular end pointc Q2 1.02 (0.80 to 1.31) 0.85 1.14 (0.90 to 1.44) 0.29 0.78 (0.61 to 1.00) 0.05 0.94 (0.74 to 1.19) 0.60 Q3 0.96 (0.75 to 1.23) 0.75 0.93 (0.72 to 1.20) 0.58 0.94 (0.74 to 1.20) 0.61 0.98 (0.77 to 1.24) 0.87 Q4 1.06 (0.83 to 1.35) 0.65 0.93 (0.73 to 1.19) 0.57 0.74 (0.58 to 0.96) 0.02 0.93 (0.73 to 1.19) 0.58 Overalld 0.88 0.33 0.05 0.93 Cardiac death Q2 0.99 (0.71 to 1.39) 0.97 1.16 (0.84 to 1.62) 0.37 0.76 (0.54 to 1.07) 0.12 0.95 (0.67 to 1.35) 0.78 Q3 0.82 (0.58 to 1.18) 0.29 0.96 (0.67 to 1.31) 0.80 0.84 (0.59 to 1.18) 0.31 1.21 (0.87 to 1.70) 0.26 Q4 1.03 (0.74 to 1.43) 0.88 0.92 (0.65 to 1.31) 0.65 0.64 (0.45 to 0.93) 0.02 0.85 (0.59 to 1.23) 0.39 Overall 0.62 0.54 0.11 0.21 Sudden cardiac death Q2 0.92 (0.59 to 1.43) 0.71 0.86 (0.55 to 1.34) 0.50 0.60 (0.39 to 0.94) 0.02 0.95 (0.60 to 1.50) 0.82 Q3 0.78 (0.49 to 1.25) 0.30 0.99 (0.63 to 1.55) 0.98 0.69 (0.44 to 1.07) 0.10 1.23 (0.79 to 1.90) 0.36 Q4 1.13 (0.74 to 1.73) 0.56 0.95 (0.61 to 1.47) 0.82 0.61 (0.39 to 0.97) 0.04 0.84 (0.52 to 1.35) 0.46 Overall 0.43 0.91 0.09 0.36 Nonfatal MI Q2 1.16 (0.74 to 1.82) 0.51 1.13 (0.73 to 1.75) 0.58 0.82 (0.51 to 1.32) 0.42 0.92 (0.57 to 1.47) 0.72 Q3 1.17 (0.75 to 1.83) 0.49 1.01 (0.64 to 1.60) 0.96 1.12 (0.71 to 1.76) 0.64 1.01 (0.64 to 1.60) 0.97 Q4 1.11 (0.71 to 1.74) 0.65 0.98 (0.63 to 1.52) 0.91 0.84 (0.53 to 1.35) 0.48 1.25 (0.81 to 1.93) 0.32 Overall 0.90 0.91 0.42 0.54 All cardiac events Q2 1.21 (0.96 to 1.52) 0.10 1.36 (1.09 to 1.71) 0.01 0.83 (0.65 to 1.05) 0.13 0.86 (0.68 to 1.10) 0.24 Q3 1.02 (0.80 to 1.30) 0.89 1.07 (0.84 to 1.37) 0.59 0.94 (0.74 to 1.19) 0.61 1.06 (0.84 to 1.34) 0.61 Q4 1.10 (0.87 to 1.39) 0.42 0.98 (0.77 to 1.25) 0.88 0.73 (0.57 to 0.94) 0.02 0.94 (0.74 to 1.19) 0.60 Overall 0.33 0.01 0.01 0.34 All cerebrovascular events Q2 0.98 (0.63 to 1.52) 0.91 0.96 (0.62 to 1.47) 0.84 0.99 (0.64 to 1.53) 0.95 0.86 (0.57 to 1.29) 0.46 Q3 1.05 (0.68 to 1.62) 0.82 0.79 (0.50 to 1.24) 0.31 0.83 (0.52 to 1.32) 0.43 0.65 (0.42 to 1.07) 0.05 Q4 1.06 (0.69 to 1.62) 0.80 1.01 (0.67 to 1.53) 0.96 0.91 (0.58 to 1.42) 0.68 0.76 (0.49 to 1.17) 0.22 Overall 0.98 0.68 0.84 0.27 Death from all causes Q2 0.93 (0.74 to 1.16) 0.50 0.97 (0.77 to 1.21) 0.78 0.77 (0.62 to 0.96) 0.02 0.84 (0.67 to 1.05) 0.13 Q3 0.79 (0.63 to 0.99) 0.04 0.95 (0.76 to 1.20) 0.68 0.75 (0.59 to 0.94) 0.01 0.93 (0.74 to 1.16) 0.50 Q4 0.94 (0.76 to 1.17) 0.60 0.89(0.71 to 1.20) 0.32 0.63 (0.49 to 0.80) ,0.001 0.78 (0.61 to 0.98) 0.04 Overall 0.23 0.80 0.002 0.15 HR, hazard ratio; 95% CI, 95% confidence interval; Q, quartile; MI, myocardial infarction. aHR calculated with multivariate Andersen–Gill model; group of patients within the first quarter of the distribution used as the reference group; atorvastatin and placebo group pooled; multiple events considered. bP value of the Wald test statistic. cCombined vascular end point consisting of MI, cardiac death, and stroke. dP value of the multivariate Wald test statistic investigating whether there was any difference between any two quartiles.

This study has several strengths. First, the study participants and propensity score–matched subgroup analyses for apoA2. underwent very detailed clinical and laboratory examinations. Fifth, the sample size of the cohort including .1000 patients Second, there was a precise follow-up for all-cause mortality on maintenance hemodialysis treatment was very large. and various vascular end points. Third, laboratory testing was One limitation of these analyses is that they were secondary, repeated after 4 weeks and then every 6 months. In contrast exploratory, and thus not predefined. Consequently, the with previous studies, this enabled us to control for longitu- results, especially those for apoA2, require confirmation in dinal measurements of HDL cholesterol during the follow-up. future studies. We also do not present data on HDL function- Fourth, we performed very detailed statistical analyses of the ality. However, previous mechanistic studies addressed this associations of HDL cholesterol and apolipoproteins with issue in detail.10–12 HDL cholesterol levels were not considered outcomes including stratified analyses, cubic spline modeling, for the decision to include or exclude a patient. Nevertheless,

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Figure 1. No association of HDL cholesterol with end points. Figure 2. Trend toward inverse relationship of apoA2 with death Spline fit of the association between HDL cholesterol and the from any cause. Spline fit of the association between apoA2 and primary end point (A) and all-cause death (B). HR, hazard ratio. CB, the primary end point (A) and all-cause death (B). HR, hazard ratio; confidence band. CB, confidence band.

implicit confounding conferred by the use of exclusion criteria blood sample was taken and information about any suspected (e.g., exclusion of several outliers for LDL cholesterol) cannot end point or serious adverse event was obtained. The study was ap- be totally ruled out.13 proved by the local ethics committee and performed in accordance In summary, HDL cholesterol does not predict all-cause with the Declaration of Helsinki. Informed consent was obtained mortality and vascular complications in patients on mainte- from all participants. nance hemodialysis treatment. A possible beneficial effect of apoA2 on survival will require confirmation in future studies. Outcome Measures The 4D study end points were centrally adjudicated by three mem- bers of the end point committee blinded to study treatment and CONCISE METHODS according to predefined criteria. The primary end point of the 4D study was defined as a composite of cardiac death, nonfatal Study Design, Setting, and Participants myocardial infarction, and stroke, whichever occurred first (com- The design of the 4D study was previously reported in detail.13,14 bined cardiovascular events). Sudden cardiac death was considered Briefly, the 4D study is a prospective, randomized, multicenter trial as previously described.13,14 Myocardial infarction was diagnosed including 1255 patients with type 2 diabetes mellitus who were aged when at least two of three criteria were met, including typical symp- 18–80 years and were on hemodialysis for ,2 years. Recruitment toms, elevated levels of cardiac , or diagnostic changes in started in March 1998 and ended in October 2002. Patients were the electrocardiogram. Stroke was defined as a neurologic deficit randomly assigned to receive double-blinded treatment with either lasting longer than 24 hours. Computed tomographic or magnetic 20 mg of atorvastatin (n= 619) or placebo (n=636) once daily. The resonance imaging results were available in all but 16 patients. participants were reexamined at 4 weeks and then every 6 months We evaluated the following end points in this analysis: combined after randomization until March 2004. At each follow-up visit, a primary end point, cardiac death, sudden cardiac death, nonfatal

490 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 484–492, 2015 www.jasn.org CLINICAL RESEARCH myocardialinfarction,allcardiacevents combined, all cerebrovascular algorithm without Caliper implemented in the STATA modul events, and all-cause death.14 psmatch2. Nonmatched individuals were excluded from the reanal- ysis. Finally, we calculated a Cox model for the association of apoA2 Laboratory Procedures as a standardized continuous variable with all-cause mortality (haz- The standard laboratory procedures have been performed in identical ard ratio refers to the change in hazard per 1-SD increase of apoA2). fashion as previously described.13,14 HDLcholesterolwasmeasuredwith All statistical tests were two sided. P values were corrected for multiple the homogenous assay from Roche Diagnostics (Mannheim, Germany). testing (4 parameters37 end points=28) according to the Bonferroni were separated by a combined ultracentrifugation- correction. Thus, P values ,0.002 (0.05/28) were considered signifi- precipitation method (b quantification). Cholesterol was measured cant. All statistical analyses were conducted using the Stata Statistical with enzymatic reagents from WAKO (Neuss, Germany) on a Software package (release 12; StataCorp., College Station, TX). WAKO R30 or Olympus AU640 analyzer. We measured apoA1, apoA2, and apoC3 by turbidimetry with reagents from Greiner (Flacht, Germany). ACKNOWLEDGMENTS

Statistical Analyses The 4D study was supported by Pfizer Inc. This work was also sup- Quartiles of HDL (subgroup analyses within the fourth HDL quartile ported by the German Federal Ministry of Education and Research for patients with HDL cholesterol #50 mg/dl and .50 mg/dl) cho- (BMBF01 EO1004). lesterol and apoA1, apoA2, and apoC3 were formed. The baseline Parts of this article were orally presented in the late-breaking clini- characteristics were presented for the quartiles of the aforementioned cal trials session at the 50th European Renal Association–European parameters. Means and SDs or medians with interquartile ranges Dialysis and Transplant Association Congress, held May 18–21, 2013, were presented for continuous variables and frequencies with per- in Istanbul, Turkey. centages for categorical variables. P values for differences in the base- line characteristics across the quartiles were calculated with analysis of covariance for continuous variables and logistic regression for DISCLOSURES categorical variables. All analyses were adjusted for age and sex. An fi extended Cox regression approach (Andersen–Gill model, which al- W.M. received consulting fees, lecture fees, and research grants from P zer. fi lows adjustment for multiple events) was used to evaluate the prog- B.G. received grant support from P zer for post hoc analyses of the original 4D study. Synlab offers testing for HDL cholesterol and apolipoproteins. nostic effect of HDL cholesterol (four quartiles and subgroup analysis within the fourth quartile) and apolipoproteins (four quartiles) on 28 fi cardiovascular events and mortality. Hazard ratios (95% con dence REFERENCES intervals) and P values adjusted for all potential confounding varia- bles were calculated across quartiles of HDL cholesterol and apoli- 1. 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