The Clinical Value of HDL Function Measurements Ebtehaj, Sanam
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3
The Predictive Value of the
Antioxidative Function of HDL for Cardiovascular Disease and Graft
Failure in Renal Transplant Recipients
Lynn J. Leberkühne*1, Sanam Ebtehaj*1, Lidiya G. Dimova1, Arne Dikkers1, Robin P.F. Dullaart2, Stephan J.L. Bakker3, and Uwe J.F. Tietge1
1-Department of Pediatrics, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713GZ Groningen, The Netherlands
2-Department of Endocrinology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713GZ Groningen, The Netherlands
3-Department of Internal Medicine, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9713GZ Groningen, The Netherlands
* These authors contributed equally to this study
Atherosclerosis. 2016 Jun; 249:181-5
Abstract
Background. Protection of low-density lipoproteins (LDL) against oxidative modification is a key anti-atherosclerotic property of high-density lipoproteins (HDL). This study evaluated the predictive value of the HDL antioxidative function for cardiovascular mortality, all-cause mortality and chronic graft failure in renal transplant recipients (RTR). Methods. The capacity of HDL to inhibit native LDL oxidation was determined in vitro in a prospective cohort of renal transplant recipients (RTR, n=495, median follow-up 7.0 years). Results. The HDL antioxidative functionality was significantly higher in patients experiencing graft failure (57.4±9.7%) than in those without (54.2±11.3%; P=0.039), while there were no differences for cardiovascular and all-cause mortality. Specifically, glomerular filtration rate (P=0.001) and C-reactive protein levels (P=0.006) associated independently with antioxidative functionality in multivariate linear regression analyses. Cox regression analysis demonstrated a significant relationship between antioxidative functionality of HDL and graft failure in age-adjusted analyses, but significance was lost following adjustment for baseline kidney function and inflammatory load. No significant association was found between HDL antioxidative functionality and cardiovascular and all-cause mortality. Conclusion. This study demonstrates that the antioxidative function of HDL (i) does not predict cardiovascular or all-cause mortality in RTR, but (ii) conceivably contributes to the development of graft failure, however, not independent of baseline kidney function and inflammatory load.
1. Introduction
High density lipoprotein particles possess a number of anti-atherosclerotic functionalities such as promoting cholesterol efflux from macrophage foam cells or protecting low density lipoproteins (LDL) against oxidative modification 1-3, a major initiating factor in the process of atherosclerotic lesion development 4. Indeed, large epidemiological studies in the general population demonstrated that plasma levels of HDL cholesterol (HDL-C) can serve as a biomarker for the atheroprotective potential of HDL 5, 6. However, recent genetic studies as well as pharmacological intervention trials indicated that HDL-C levels as such do not uniformly predict cardiovascular disease (CVD) risk 2. The focus in the cardiovascular field is therefore currently shifting from HDL-C quantity determinations to efforts to measure the quality of HDL, i.e. the functional properties of these lipoproteins 1, 2, 7.
Renal transplant recipients (RTRs) have an increased risk of atherosclerosis formation, resulting in a 4-6 fold higher CVD risk 8. In addition, transplant vasculopathy, an atherosclerotic process of the vasculature of the transplanted kidney is a major cause for chronic renal transplant dysfunction, leading to graft failure 9, 10. This is a highly relevant clinical issue, since 5 years after transplantation about 50% of the patients have developed transplant vasculopathy, a number even increasing to 90% after 10 years 9. However, especially in this group of patients, classical risk factors including HDL-C concentrations do not fully explain the increase in atherosclerosis risk 9, making it likely that changes in functional properties of HDL critically contribute.
Therefore, the aim of this study was to evaluate the predictive value of a selected anti- atherosclerotic key function of HDL, namely its antioxidative potential, for the risk of future CVD mortality, all-cause mortality and graft failure in a well-characterized cohort of renal transplant recipients. Our data demonstrate that a contribution of the anti-oxidative function of HDL especially to the development of graft failure is conceivable, however, not independent of baseline kidney function and inflammatory load.
2. Materials and Methods
For an extended description please see online supplement.
2.1. Study Design and Study Population
For this prospective study we used an established patient cohort of adult renal transplant recipients from the outpatient clinic of the UMCG who have visited the hospital between August 2001 and July 2003 11, 12. The study was approved by the local Medical Ethics Committee (METc2001/039) and written informed consent was obtained from all participants. All patients had a functioning renal graft with no complications for at least 1 year to exclude early immune- mediated rejection as a potential confounding factor. Patients with other comorbidities such as endocrine abnormalities other than diabetes, congestive heart failure or malignant disease were excluded. All relevant patient characteristics were collected from the “Groningen Renal Transplant Database”, medical records and with help of a self-report questionnaire. Of the 847 eligible patients, 606 volunteered to participate and were included in the cohort. Plasma samples were available from 517 patients, of which 22 were excluded due to acute inflammation at the time of blood sampling as evidenced by high CRP levels (above 15 mg/l), leaving 495, in which the antioxidative functionality of HDL was determined. In an alternative
set of analyses (please see online supplement) patients with CRP levels above 10 mg/l were excluded (n=65), leaving n=454 patients for analysis.
2.2. Isolation of HDL and Measurement of Antioxidative Functionality
To determine HDL-mediated protection against LDL oxidation, a previously published method was used 13. Briefly, HDL was isolated by precipitation of apoB-containing lipoproteins as described 13, 14. For the anti-oxidation assay 2% of individual HDL preparations were added to native, unoxidized LDL particles (100mg/dl final protein concentration), after which oxidation was induced by 6.3μl of 2.5mM AAPH (2,2ʹ-azobis [2-amidinopropane] dihydrochloride) followed by incubation at 37°C for 10 h. After that thiobarbituric acid reactive substances (TBARS) were determined as a measure for the degree of LDL oxidation as detailed previously
13. The HDL antioxidative capacity was calculated as the percent reduction in TBARS formation obtained with an individual HDL sample relative to a reaction to which only LDL and AAPH but no HDL had been added 13. Higher values thus indicate better protection against LDL oxidation.
The antioxidation assay for all samples was carried out at the same time and with the same badges of reagents to limit potential variation due to different assay conditions. We also compared apoB-depleted plasma with (i) respective lipoprotein-free plasma preparations (supplemental figure I) and (ii) HDL isolated by sequential density ultracentrifugation (supplemental figures II and III). These combined data indicate that with the sample preparation method used in our study the anti-oxidative function is to a substantial part due to the presence of HDL. The storage time of the samples was not statistically associated with the results of the anti-oxidative function assay indicating that different length of storage is not a confounding factor.
2.3. Primary End Points
The main outcome measure of this study is the antioxidative functionality of HDL, the primary end points are cardiovascular mortality, all-cause mortality, and graft failure. Graft failure was defined as return to dialysis therapy or retransplantation. A surveillance system ensured information on patient status and cause of death between inclusion and up to the year 2009
12.
2.4. Statistical Analysis
All statistical analyses were conducted using SPSS 20. Normally distributed continuous variables are given as mean ± standard deviation, continuous variables with a skewed distribution as median [25th-75th percentile] and categorical variables by absolute numbers (percentages). Overall trends between antioxidative functionality and the three primary endpoints were tested for significant differences using student’s t-test. Renal transplant recipients were divided into tertiles according to the antioxidative functionality of HDL (low, medium, and high) and stratified for gender. Baseline characteristics of the patients were analyzed and tested for differences among these three groups. One-way analysis of variance followed by Bonferroni post hoc test was used for normally distributed variables and the Kruskal-Wallis test followed by Mann-Whitney U test for variables with a skewed distribution.
The Chi-square test was used to compare categorical data. Multivariate linear regression analyses were performed with inclusion of parameters with a P-value ≤0.1 between the tertiles
of HDL antioxidative capacity into the model and by eliminating by step-by-step backward regression (P≤0.05). Subsequently, multivariate Cox regression analysis models were created to estimate the HRs and 95% CIs for all-cause mortality, cardiovascular mortality and graft failure. Hazard ratios (HRs) are reported per 1-SD increase with 95% confidence intervals (CIs).
Power calculations indicated that the minimum detectable HR based on an assumption of 90%
power and a two-sided alpha significance of 0.05 was 0.77 for CVD mortality, 0.83 for overall mortality, and 0.75 for graft failure. A two-sided P-value of ≤0.05 was considered statistically significant.
3. Results
In this study the predictive value of HDL antioxidative functionality on the survival of patients as well as their kidney grafts was analyzed in 495 renal transplant recipients (mean age 51.6±12.0; 54% male). Patients had a median follow-up of 7.0 years [6.3–7.5 years]. Within this period, 102 (21%) patients died, which was attributable to CVD in 54 patients (11%). A total of 46 (9%) patients experienced renal graft failure. First, the antioxidative functionality at baseline was compared between patients with or without subsequent cardiovascular death, all-cause mortality and graft failure during follow-up. At baseline, patients who died due to cardiovascular causes did not have a significantly different antioxidative function (56.0±9.7%) compared to those not experiencing cardiovascular death (54.3±11.4%; P=0.236). Further, in patients who died due to any cause, antioxidative functionality was also not significantly different (56.1±9.1%) compared to those who survived (54.0±11.6%; P=0.095). However, renal transplant recipients who did not experience graft failure had a significantly lower antioxidative HDL function (54.2±11.3%), compared to those with subsequent graft failure (57.4±9.7%;
P=0.039). When using a cut-off value for CRP of 10 mg/l, these differences were no longer stiatistically significant (please see online supplement for details). Next, baseline characteristics of the patients were analyzed. For this purpose, patients were divided into tertiles, based on their HDL antioxidative functionality stratified for gender: low 44.0% [39.4-48.9], medium 55.7% [53.2-57.9] and high 64.4% [61.5-67.6]. Table 1 shows the relevant baseline parameters, for the complete data set see online supplement (Supplemental Table I).
Table 1: Most relevant characteristics of renal transplant recipients divided into tertiles based on HDL antioxidative functionality and stratified for gender.
*Tertile significantly different from first (low) tertile, P <0.05; #Tertile significantly different from first (low) tertile, P
<0.01; §Tertile significantly different from second (medium) tertile, P <0.05; ‡Tertile significantly different from second (medium) tertile, P <0.01. Data are given either as mean ± SD or median [25th-75th percentile].
Age of the renal transplant recipients was not found to differ according to antioxidative functionality. Patients with higher antioxidative capacity had higher plasma levels of total cholesterol (P=0.035) and triglycerides (P=0.006). Apolipoprotein A-I, a major protein component of HDL, was inversely related to antioxidative function (P=0.044). Higher levels of hsCRP, thus higher inflammatory load, were found to be associated with better antioxidative capacities of HDL (P=0.006). Differences in insulin concentration and insulin resistance (HOMA- IR) among the different groups divided according to antioxidative functionality were also detected. In addition, better anti-oxidative function was associated with lower glomerular filtration rate (eGFR), thus decreased kidney function. Then multivariate linear regression analyses were performed to assess which variables are independently associated with antioxidative functionality of HDL. All parameters found to show a difference of P<0.10 between the tertiles of HDL antioxidative capacity, were included in the analysis (Table 2).
Table 2: Variables independently associated with antioxidative functionality of HDL.
Beta 95% CI Standardized beta P Value
eGFR, mL/min/1.73m2 -0.103 [-0.164; -0.042] -0.146 0.001
Male gender, n (%) 3.083 [1.150; 5.017] 0.137 0.002
hsCRP, mg/L 2.621 [0.739; 4.504] 0.121 0.006
Insulin, mmol/L -5.655 [-9.838; -1.472] -0.116 0.008
Variables are listed in decreasing order of strength of association according to the value of the standardized beta.
Parameters
Tertiles according to Antioxidative Functionality
P value
Low Medium High
HDL Antiox. Function (%) 44.0 [39.4-48.9] 55.7 [53.2-57.9]# 64.4 [61.5-67.6]#,‡ <0.001 Age of patient, years 52.7 [43.8-60.2] 53.8 [42.3-61.1] 52.8 [44.1-59.9] 0.705
Male gender, n (%) 90 (54.5) 90 (54.2) 89 (54.3) 1.000
BMI, kg/m2 25.7 ± 4.4 25.8 ± 4.0 26.3 ± 4.3 0.469
Total Cholesterol, mmol/L 5.5 ± 1.0 5.6 ± 0.9 5.8 ± 1.2 0.035
Apolipoprotein A-I, g/L 1.6 ± 0.3 1.6 ± 0.3 1.5 ±0.3§ 0.044
LDL Cholesterol, mmol/L 3.4 ± 1.0 3.6 ± 0.9 3.7 ± 1.1 0.098
HDL Cholesterol, mmol/L 1.1 ± 0.3 1.1 ± 0.3 1.1 ± 0.3 0.125
Triglycerides, mmol/L 1.8 [1.3-2.6] 1.8 [1.4-2.3] 2.1 [1.5-2.9]*, ‡ 0.006
Insulin, mmol/L 12.1 [8.7-17.9] 10.1 [7.7-12.9] # 10.9 [7.7-14.8]* 0.003
HOMA-IR 2.4 [1.7-4.0] 2.0 [1.5-2.9] # 2.3 [1.5-3.2] 0.006
hsCRP, mg/L 1.5 [0.6-3.4] 2.0 [0.9-4.4]* 2.2 [1.1-5.3]# 0.006
eGFR, mL/min/1.73m2 49.2 ± 16.6 48.9 ±15.4 43.7 ± 14.9#,‡ 0.002
Antioxidative functionality had an independent association with eGFR, gender, plasma hsCRP and insulin concentration, however, the R2 of the final model was 0.07.
Further, multivariate Cox regression analysis was carried out to obtain proportional hazard ratios and evaluate the independent contribution of antioxidative functionality to the risk of mortality and graft failure (Table 3). Antioxidative function of HDL did not have a significant relationship with all-cause (P=0.108) or specific cardiovascular mortality (P=0.276), a finding remaining unchanged after correction for age, gender, eGFR and CRP. On the other hand, antioxidative capacity was found to be significantly associated with the risk of graft failure (P=0.038). After adjustment for age, this association did not change appreciably (P=0.035).
After additional adjustment for gender, this association was still strong, but lost formal statistical significance (P=0.056). Following further adjustment for eGFR, and CRP the association with graft failure is lost. Taken together, these results indicate that the antioxidative functionality of HDL might contribute to the development of graft failure in RTR, however, not independent of eGFR and CRP.
Table 3: Hazard ratios for graft failure, cardiovascular mortality and all-cause mortality by antioxidative functionality of HDL.
Graft Failure Cardiovascular Mortality All-cause Mortality
(46 events) (54 events) (102 events)
HR [95%CI]
per 1-SD increase
P Value HR [95%CI]
per 1-SD increase
P Value HR [95%CI]
per 1-SD increase P Value Model 1
(crude)
1.03
[1.00-1.06] 0.038* 1.01
[0.99-1.04] 0.276 1.02
[1.00-1.03] 0.108 Model 2
(model 1 + age)
1.03
[1.00-1.06] 0.035* 1.01
[0.99-1.04] 0.300 1.02
[1.00-1.03] 0.123 Model 3
(model 2 + gender)
1.03
[1.00-1.96] 0.056 1.01
[0.99-1.04] 0.314 1.02
[1.00-1.03] 0.128 Model 4
(model 2 + eGFR)
1.01
[0.98-1.04] 0.571 1.01
[0.98-1.03] 0.660 1.01
[0.99-1.03] 0.392 Model 5
(model 3 + hsCRP)
1.01
[0.98-1.04] 0.535 1.00
[0.98-1.03] 0.832 1.01
[0.99-1.03] 0.514
*statistically significant, P <0.05.
4. Discussion
The results of this prospective study suggest that, at least in RTR, the antioxidative functionality of HDL might not have the potential to serve as a clinically relevant predictive biomarker for cardiovascular disease, all-cause mortality or the development of chronic graft failure. RTR were chosen, since in this patient group the pathophysiological mechanisms of CVD but also of chronic atherosclerosis-mediated graft failure are not well defined, however, traditional risk
factors and specifically HDL-C cannot fully explain the increased risk 9, 15, 16. In our reasoning, these observations increased the probability for changes in HDL function to have an impact.
The most interesting finding of our study is that baseline antioxidative functionality predicts graft failure in RTR, at least in crude and age-adjusted models. Unexpectedly though, a better antioxidative HDL function was associated with a higher risk of graft failure. A potential explanation could be derived from the observation that this correlation was lost once either an even stricter cut-off value for plasma CRP was used (10 mg/l instead of 15 mg/l, for data please see online supplement) or after correcting for potential confounders, most importantly eGFR and CRP. Both, a decline in kidney function and an increased inflammatory load are conditions of increased in vivo oxidative stress 17, 18. It is primarily counterintuitive that higher oxidative stress is associated with better anti-oxidative function of HDL. However, this association could be conceivably caused by a compensatory response namely an attempt to increase antioxidative defense mechanisms to meet the increased demand as e.g. has been shown in smokers 19. That this attempt remains unsuccessful could be due to a decrease in other HDL functions in proinflammatory conditions, such as cholesterol efflux and reverse cholesterol transport 2, 20. The plausibility of these considerations is clinically supported by a study showing that high CRP levels offset the tight inverse correlation between HDL-C and risk of atherosclerotic cardiovascular disease; the authors also interpreted their findings as indicative of inflammation decreasing the atheroprotective functionality of HDL 21. However, to formally prove this point further studies are required. Also, it should be noted that an absolute number of 46 patients with graft failure is relatively low for statistical analysis; therefore, although in itself robust, the results should be treated with caution.
Another potential impacting factor could be LCAT. LCAT is an enzyme, which is associated with HDL and is involved in modulating LDL oxidation 13, 22. We concluded from a previous study that increased LCAT activity contributes to an impaired antioxidative functionality of HDL 13. The activity of LCAT itself is reduced with impaired kidney function or inflammation, both present in RTR 23, 24. In this scenario a reduced LCAT activity might result in improved antioxidative functionality of the HDL particle. However, LCAT levels were not determined in the present study.
Furthermore, the anti-oxidative function of HDL could be influenced by the administered medication. In our study a better HDL function tended to be associated with the number of anti-hypertensives as well as the use of calcineurin inhibitors. Although in the present work for the anti-hypertensive medication this trend was not confirmed for individual classes of drugs, previous studies suggested that at least β-blockers, angiotensin converting enzyme inhibitors and angiotensin II receptor 1 antagonists can reduce oxidative stress and lipid peroxidation 25,
26. Calcineurin inhibitors on the other hand are rather thought to induce oxidative stress 27-29. Taken together, although the association did not reach the level of statistical significance, it can not be excluded at present that medication contributes to the observations made in our study and, in a broader perspective, to findings in cohorts of RTR in general.
Another finding of our study worthwhile to point out is the U-shaped relationship between the HDL anti-oxidative function and HOMA-IR. Since blood glucose values (supplemental table I) did not differ between the different tertiles of HDL anti-oxidative functionality, this relationship seems primarily due to differences in circulating insulin levels. HDL has been shown to impact both, insulin secretion by pancreatic β-cells 30 as well as glucose uptake in the periphery by
skeletal muscle 31. However, the current study was not designed to specifically investigate a relationship between HDL function and parameters of glucose metabolism; therefore, a potential causality of our observation as well as the delineation of impacting factors remain unresolved. Future studies with a specific focus on glucose metabolism parameters would be required to clarify these points.
In general, studies addressing the prospective value of HDL function measurements on clinically relevant outcomes are scarce. There are thus far only two reports evaluating a different functionality of HDL, namely cholesterol efflux, in predicting future cardiovascular mortality in the general population. Interestingly, in one the authors found, also against expectations, that a high cholesterol efflux capacity was associated with an increased risk for CVD and death 32, while in the other better efflux at baseline predicted lower CVD risk during follow-up 33. Clearly, more research is needed to understand the complex relationship between factors determining HDL quality independent of HDL-C levels and the clinical significance of HDL function in different relevant settings, including changes in response to therapeutic intervention. It needs to be taken into account for the interpretation of HDL function studies that no gold standards are available for respective assays and not even for methods to isolate HDL 2. Therefore, interpretation of each individual study depends on the assay conditions applied, the respective experimental read-outs and the methodology to isolate HDL, which are complex particles differing in size and carrying a diverse lipid and protein cargo 2. In the present study we decided to use plasma after precipitation of apoB-containing lipoproteins, similar to studies by other groups addressing HDL anti-oxidative properties 14. This method is suitable for large numbers of clinical samples, however, also leaves other components of plasma. At present we can not, based on the design of our study, formally exclude that these might contribute to the associations found in our analyses.
In conclusion, our study shows that the antioxidative functionality of HDL might not be a clinically relevant parameter to predict future cardiovascular or all-cause mortality in RTR as well as chronic graft failure. Graft failure was predicted in these patients to a certain extent, however, not independent of baseline kidney function and inflammatory load, and in a fashion that might be opposite to expectations. More research, also including other biologically relevant functions of HDL will be required to delineate the prospective value of HDL function for CVD and graft failure in RTR.
Acknowledgements
This study was supported by grants from the Netherlands Organization for Scientific Research (VIDI Grant 917-56-358 to U.J.F.T.), the Top Institute (TI) Food and Nutrition (to U.J.F.T.), and the Dutch Kidney Foundation (Nierstichting Nederland C00.1877 to S.J.L.B.).
Supplement
Supplemental Materials and Methods
Isolation of HDL
Venous EDTA plasma samples of the patients were available for this study. In order to avoid the influence of VLDL and LDL present in these samples, apoB-containing lipoproteins were precipitated by adding 75μL of polyethylene glycol 6000 in 10mM HEPES (pH 8) to 150μL of plasma followed by mixing and incubating on ice for 30 min 1,2. Subsequently, the samples were centrifuged (2200g at 4°C) for 30min. Supernatants were transferred to new tubes, which were then used for the current study.
Measurement of Antioxidative Functionality
For the anti-oxidation assay 2.5μl of the individual isolated apoB-depleted plasma preparations containing the patients HDL were added to vials containing 96.4μl PBS, 19.8μl native, unoxidized LDL (100mg/dl protein) and 6.3μl 2.5mM AAPH (2,2ʹ-azobis [2-amidinopropane]
dihydrochloride) resulting in a 2% HDL solution. The samples were incubated for 10 hours at 37°C to induce LDL oxidation. Subsequently, 100μl of the samples were transferred into a new Eppendorf tube, containing 200μl ice-cold 10% trichloroacetic acid (TCA) for protein precipitation. The samples were incubated for 15 minutes on ice and then centrifuged for 15 minutes at 2200g at 4°C. Afterwards, 200μl of the supernatant was transferred to clean 1.5 ml Eppendorf tube, containing 200μl of 0.67% thiobarbituric acid (TBA). The tubes were incubated 10 minutes at 99°C in a thermomixer. After cooling down samples were added in duplicate to a black 96-well plate and fluorescence at 510nm excitation and 553nm emission was measured.
The fluorescence of the thiobarbituric acid reactive substances (TBARS) was used as a measure for the degree of LDL oxidation. Individual values were determined with the means of a standard curve. Background values of PBS+AAPH preparations without lipoproteins were subtracted from individual data. Incubations with AAPH and LDL but without HDL gave maximal oxidation values. In addition, two healthy control samples were included on each plate to check and potentially correct for plate-to-plate variation. The HDL antioxidative capacity was then calculated as the difference between the maximal amount of TBARS formed in a reaction to which no HDL had been added and the percent reduction obtained with an individual HDL sample. Higher values indicate better protection against oxidation, and thus a higher antioxidative functionality. The antioxidation assay for all samples was carried out at the same time and with the same reagents to limit potential variation due to different assay conditions.
Measurements and Definitions
The definition of the characteristics of the database of the renal transplant recipients as well as the routine laboratory methods used have been described previously in detail 3,4. In summary, blood was drawn after an 8-12 hours overnight fasting period and then total and HDL cholesterol were measured using the cholesterol oxidase-phenol aminophenazone method.
Apolipoprotein A-I was measured by immunoturbidimetry. Using the Friedewald equation, LDL cholesterol was calculated. Plasma triglycerides were determined by the glycerol-3-phosphate
oxidase-phenol aminophenazone method. Blood pressure was measured using an automatic device (Omron M4; Omron Europe B.V., The Netherlands) in supine position after 6 minutes rest as the average of three measurements at 1-minute intervals. Body mass index was calculated by dividing weight (kg) by height squared (m2). Plasma insulin was assessed using an AxSym autoanalyzer. Insulin resistance was calculated using Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Plasma high-sensitivity (hs)CRP was measured by ELISA.
Glomerular filtration rate (eGFR) was estimated by using creatinine clearance, calculated from 24-hour urinary creatinine excretion and plasma creatinine. With the Biuret reaction, total urinary protein concentration was analyzed and a urinary protein excretion ≥ 0.5g per 24 hours was defined as proteinuria.
Supplemental Results
Below is a description of the re-analysis of the data set after additionally also excluding all patients with a CRP level above 10mg/l. In this study the predictive value of HDL antioxidative functionality on the survival of patients as well as their kidney grafts was analyzed in 454 renal transplant recipients (mean age 51.3±11.9; 54,8% male). Patients had a median follow-up of 7.0 years [6.3–7.5 years]. Within this period, 86 (18.9%) patients died, which was attributable to CVD in 44 (9.7%) patients. A total of 40 (8.8%) patients experienced renal graft failure. First, the antioxidative functionality at baseline was compared between patients with or without subsequent cardiovascular death, all-cause mortality and graft failure during follow-up. At baseline, patients who died due to cardiovascular causes did not have a significantly different antioxidative function (56.4±10.3%) compared to those not experiencing cardiovascular death (53.9±11.4%; P=0.181). Further, in patients who died due to any cause, antioxidative functionality was also not significantly different (56.2±9.2%) compared to those who survived (53.7±11.7%; P=0.064). In addition, also in renal transplant recipients who experienced graft failure the antioxidative HDL function was not statisticall different, compared to those with subsequent graft failure (56.9±10.1% vs. 53.9±11.4%; P=0.118,). Next, baseline characteristics of the patients were analyzed. For this purpose, patients were divided into tertiles, based on their HDL antioxidative functionality stratified for gender: low (n=152; 43.9% [39.3-48.8]), medium (n=150; 55.2% [52.6-57.3]) and high (n=152; 64.3% [61.4-67.5). Supplemental table II shows the relevant baseline parameters. Age of the renal transplant recipients was not found to differ according to antioxidative functionality. Patients with higher antioxidative capacity had higher plasma levels of total cholesterol (P=0.039) and triglycerides (P=0.004).
Apolipoprotein A-I, a major protein component of HDL, was inversely related to antioxidative function (P=0.029). Higher levels of hsCRP, thus higher inflammatory load, were found to be borderline significantly associated with better antioxidative capacities of HDL (P=0.063).
Differences in insulin concentration and insulin resistance (HOMA-IR) among the different groups divided according to antioxidative functionality were also detected. In addition, better anti-oxidative function was associated with lower glomerular filtration rate (eGFR), thus decreased kidney function.
Then multivariate linear regression analyses were performed to assess which variables are independently associated with antioxidative functionality of HDL. All parameters found to show a difference of P<0.10 between the tertiles of HDL antioxidative capacity, were included in the analysis (Supplemental table III). Antioxidative functionality had an independent association with eGFR, gender, plasma hsCRP and insulin concentration, however, the R2 of the final model was 0.06.
Further, multivariate Cox regression analysis was carried out to obtain proportional hazard ratios and evaluate the independent contribution of antioxidative functionality to the risk of mortality and graft failure (Supplemental table IV). Antioxidative function of HDL did not have a significant relationship with all-cause (P=0.074) or specific cardiovascular mortality (P=0.174) and neither with graft failure (P=0.071), findings that remained unchanged after correction for age, gender, eGFR and CRP.
References
1. Kappelle PJ, de Boer JF, Perton FG, et al., Increased LCAT activity and hyperglycaemia decrease the antioxidative functionality of HDL, Eur J Clin Invest, 2012;42:487-495.
2. Rohatgi A, Khera A, Berry JD, et al., HDL cholesterol efflux capacity and incident cardiovascular events, N Engl J Med, 2014;371:2383-2393.
3. Baia LC, Humalda JK, Vervloet MG, Navis G, Bakker SJ, de Borst MH,
Fibroblast growth factor 23 and cardiovascular mortality after kidney transplantation. Clin J Am Soc Nephrol, 2013;8:1968-1978.
4. de Vries AP, Bakker SJ, van Son WJ, et al., Metabolic syndrome is associated with impaired long- term renal allograft function; not all component criteria contribute equally, Am J Transplant, 2004;4:1675-1683.
Supplemental Tables
Supplemental table I: Complete characteristics of patients divided into tertiles based on HDL antioxidative functionality and stratified for gender.
Parameters
1st Tertile Low
2nd Tertile Medium
3rd Tertile
High P value
(n=165) (n=166) (n=164)
HDL Antioxidative Function (%) 44.0 [39.4-48.9]
55.7 [53.2-57.9]#
64.4
[61.5-67.6]#,‡ <0.001 Recipient demographics
Age, years 52.7
[43.8-60.2]
53.8 [42.3-61.1]
52.8
[44.1-59.9] 0.705
Male gender, n (%) 90 (54.5) 90 (54.2) 89 (54.3) 1.000
Current Smoking, n (%) 34 (20.6) 29 (17.5) 42 (25.6) 0.197
Previous Smoking, n (%) 71 (43.0) 72 (43.4) 70 (42.7) 0.993
Body Composition
BMI, kg/m2 25.7 ± 4.4 25.8 ± 4.0 26.3 ± 4.3 0.469
Waist circumference men, cm 99.5
[90.5-106.0]
97.5 [91.4-107.6]
102.0
[92.3-110.0] 0.286 Waist circumference women, cm 93.0
[81.0-105.0]
94.3 [82.3-101.4]
91.0
[83.5-106] 0.927
Lipids
Total Cholesterol, mmol/L 5.5 ± 1.0 5.6 ± 0.9 5.8 ± 1.2 0.035
LDL Cholesterol, mmol/L 3.4 ± 1.0 3.6 ± 0.9 3.7 ± 1.1 0.098
HDL Cholesterol, mmol/L 1.1 ± 0.3 1.1 ± 0.3 1.1 ± 0.3 0.125
Apolipoprotein A-I, g/L 1.6 ± 0.3 1.6 ± 0.3 1.5 ±0.3§ 0.044
Triglycerides, mmol/L 1.8
[1.3-2.6]
1.8 [1.4-2.3]
2.1
[1.5-2.9]*, ‡ 0.006
Use of statins, n (%) 83 (50.3) 81 (48.8) 88 (53.7) 0.657
Cardiovascular disease history
Myocardial infarction, n (%) 15 (9.2) 16 (9.8) 11 (6.7) 0.565
TIA/CVA, n (%) 9 (5.5) 8 (4.9) 8 (4.9) 0.935
Blood pressure
Systolic blood pressure, mmHg 150 ± 21.4 153.6 ± 23.7 154.3 ± 23.5 0.190
Diastolic blood pressure, mmHg 89.8 ± 9.7 89.1 ± 9.6 90.1 ± 10.6 0.623
Use of ACE inhibitors, n (%) 56 (33.9) 54 (32.5) 62 (37.8) 0.585
Use of β-blockers, n (%) 92 (55.8) 101 (60.8) 109 (66.5) 0.139
Use of diuretics, n (%) 68 (41.2) 67 (40.4) 79 (48.2) 0.297
Number of antihypertensive drugs 2 [1-3] 2 [1-3] 2 [1-3] *,§ 0.052
Glucose homeostasis
Glucose, mmol/L 4.6
[4.0-5.1]
4.5 [4.1-4.9]
4.6
[4.2-5.1] 0.238
Insulin, mmol/L 12.1
[8.7-17.9]
10.1 [7.7-12.9] #
10.9
[7.7-14.8]* 0.003
HOMA-IR 2.4
[1.7-4.0]
2.0 [1.5-2.9] #
2.3
[1.5-3.2] 0.006
Post-Tx diabetes mellitus, n (%) 27 (16.4) 31 (18.7) 31 (18.7) 0.808
Use of anti-diabetic drugs, n (%) 24 (14.5) 24 (14.5) 20 (12.2) 0.778
Use of insulin, n (%) 12 (7.3) 12 (7.2) 8 (4.9) 0.613
Inflammation
hsCRP, mg/L 1.5
[0.6-3.4] 2.0
[0.9-4.4]* 2.2
[1.1-5.3]# 0.006 Donor demographics
Age, years 38
[22-50.1]
36.5 [21-49]
40
[24-51] 0.426
Male gender, n (%) 94 (57.0) 92 (56.1) 88 (53.7) 0.822
Living kidney donor, n (%) 23 (13.9) 20 (12.0) 20 (12.2) 0.875
Postmortem donor, n (%) 142 (86.1) 146 (88.0) 144 (87.8) 0.875
(Pre-)transplant history
Dialysis time, months 24.0
[11.5-46.5]
28 [13-48.3]
29
[14.0-49.0] 0.471 Time between Tx and inclusion, years 7.3
[3.1-12.6]
5.5 [2.7-10.3]
6.0
[2.4-11.8] 0.083 Immunosuppresive medication
Daily prednisolone dose, mg/dL 10 [7.5-10.0]
10 [7.5-10.0]
10
[8.8-10.0] 0.341
Calcineurin inhibitors, n (%) 123 (74.5) 129 (77.7) 139 (84.8) 0.063
Proliferation inhibitors, n (%) 124 (75.2) 129 (77.7) 114 (69.5) 0.258
Renal allograft function
eGFR, mL/min/1.73m2 49.2 ± 16.6 48.9 ±15.4 43.7 ± 14.9#,‡ 0.002
Urinary protein excretion, g/24h 0.2 [0.0-0.5]
0.2 [0.0-0.5]
0.3
[0.1-0.5] 0.096
Proteinuria >0.5g/24h, n (%) 49 (29.7) 40 (24.1) 48 (29.3) 0.454
*Tertile significantly different from first tertile, P <0.05; #Tertile significantly different from first tertile, P <0.01; §Tertile significantly different from second tertile, P <0.05; ‡Tertile significantly different from second tertile, P <0.01.
Supplemental table II: Most relevant characteristics of renal transplant recipients divided into tertiles based on HDL antioxidative functionality and stratified for gender.
Parameters
Tertiles according to Antioxidative Functionality
P value Low
n=152
Medium n=150
High n = 152
HDL Antiox. Function (%) 43.9 [39.3-48.8] 55.2 [52.6 -57.3]# 64.3 [61.4-67.5]#,‡ <0.001 Age of patient, years 53.0 [43.3-60.3] 52.4 [42.1-60.5] 52.9 [43.6-59.9] 0.960
Total Cholesterol, mmol/L 5.6 ± 1.0 5.5 ± 0.9 5.8 ± 1.3 0.039
Apolipoprotein A-I, g/L 1.6 ± 0.3 1.6 ± 0.3 1.5 ±0.3§ 0.029
Triglycerides, mmol/L 1.8 [1.4-2.6] 1.8 [1.2-2.2] 2.0 [1.5-2.9]‡ 0.004
Insulin, mmol/L 12.2 [8.7-18.5] 10.0 [7.8-12.7]# 11.1 [7.8-15.0]* 0.002
HOMA-IR 2.5 [1.7-4.1] 2.0 [1.5-2.8]# 2.2 [1.5-3.2]* 0.003
hsCRP, mg/L 1.4 [0.6-3.1] 1.8 [0.7-3.9] 1.9 [0.8-4.2] 0.063
eGFR, mL/min/1.73m2 49.2 ± 16.5 49.7 ±15.3 44.2 ± 15.1*,‡ 0.003
Time between taking blood sample and measurement, months
140.8 ± 6.4 138.2 ± 5.9# 138.3 ± 5.0# <0.001
*Tertile significantly different from first (low) tertile, P <0.05; #Tertile significantly different from first (low) tertile, P <0.01; §Tertile significantly different from second (medium) tertile, P <0.05; ‡Tertile significantly different from second (medium) tertile, P <0.01. Data are given either as mean ± SD or median [25th-75th percentile].
Supplemental table III: Variables independently associated with antioxidative functionality of HDL.
Beta 95% CI Standardized beta P Value
Male gender 3.343 [1.315; 5.371] 0.147 0.001
eGFR, mL/min/1.73m2 -0.101 [-0.165; -0.037] -0.141 0.002
Insulin, mmol/L, log value -5.816 [-10.219; -1.413] -0.118 0.010
hsCRP, mg/L, log value 2.417 [0.251; 4.583] 0.100 0.029
Variables are listed in decreasing order of strength of association according to the value of the standardized beta.
Supplemental table IV: Hazard ratios for graft failure, cardiovascular mortality and all-cause mortality by antioxidative functionality of HDL.
Graft Failure Cardiovascular Mortality All-cause Mortality
(46 events) (54 events) (102 events)
HR [95%CI] P Value HR [95%CI] P Value HR [95%CI] P Value
Model 1 (crude)
1.03
[1.00-1.06] 0.071 1.02
[0.99-1.05] 0.174 1.02
[1.00-1.04] 0.074 Model 2
(model 1 + age)
1.03
[1.00-1.06] 0.066 1.02
[0.99-1.05] 0.175 1.02
[1.00-1.04] 0.077 Model 3
(model 2 + gender)
1.03
[1.00-1.06] 0.095 1.02
[0.99-1.05] 0.199 1.02
[1.00-1.04] 0.079 Model 4
(model 3 + eGFR)
1.01
[0.98-1.04] 0.643 1.01
[0.98-1.04] 0.432 1.01
[0.99-1.03] 0.280 Model 5
(model 4 + hsCRP)
1.01
[0.98-1.04] 0.546 1.01
[0.98-1.04] 0.445 1.01
[0.99-1.03] 0.290
*statistically significant, P <0.05.
Supplemental Figures
Supplemental figure I. Comparison of the anti-oxidative functionality of apoB-depleted plasma to lipoprotein-free plasma.
To investigate the part of the anti-oxidative activity of apolipoprotein B (apoB)-depleted plasma that was due to the presence of HDL, plasma from healthy controls (n=15) was depleted from apoB-containing lipoproteins as detailed in “Methods”, then HDL was removed by ultracentrifugation (d = 1.25 g/ml) to obtain the lipoprotein free fraction. KBr was removed by dialysis against PBS using GeBAflex-tubes (MWCO 6-8 kDa, Gene-Bio-Application L.T.D.). ***, p<0.001.
Supplemental figure II. Comparison of the anti-oxidative functionality of apoB-depleted plasma to isolated HDL in healthy controls.
To investigate the contribution of HDL to the anti-oxidative activity of apolipoprotein B (apoB)-depleted plasma, plasma from healthy controls (n=15) was depleted from apoB-containing lipoproteins as detailed in “Methods”; then HDL was isolated by ultracentrifugation (1.063 < d < 1.21 g/ml) using KBr followed by dialysis against PBS in GeBAflex-tubes (MWCO 6-8 kDa, Gene- Bio-Application L.T.D.). (A) Results given as mean±SD, ***, p<0.001. (B) Correlation between the results obtained with the two respective methods of HDL isolation.
Supplemental figure III. Comparison of the anti-oxidative functionality of apoB-depleted plasma to isolated HDL in RTRs.
To investigate the contribution of HDL to the anti-oxidative activity of apolipoprotein B (apoB)-depleted plasma, plasma from healthy controls (n=15) was depleted from apoB-containing lipoproteins as detailed in “Methods”; then HDL was isolated by ultracentrifugation (1.063 < d < 1.21 g/ml) using KBr followed by dialysis against PBS in GeBAflex-tubes (MWCO 6-8 kDa, Gene- Bio-Application L.T.D.). (A) Results given as mean±SD, **, p<0.01. (B) Correlation between the results obtained with the two respective methods of HDL isolation.
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