Article

Alteration of HDL Protein Composition with Hemodialysis Initiation

Ke Wang,1,2 Leila R. Zelnick ,1,2 Andrew N. Hoofnagle,1,2,3 Tomas Vaisar,1 Clark M. Henderson,3 Peter B. Imrey ,4,5 Cassianne Robinson-Cohen ,6 IanH.deBoer,1,2 Yan-Ting Shiu,7 Jonathan Himmelfarb,1,2 Gerald J. Beck,4,5 and Bryan Kestenbaum,1,2 on behalf of the HFM Study

Abstract Departments of Background and objectives HDL particles obtained from patients on chronic hemodialysis exhibit lower 1 fl fl Medicine and ef ux capacity and are enriched in in ammatory proteins compared with those in healthy individuals. 3Laboratory Medicine Observed alterations in HDL proteins could be due to effects of CKD, but also may be influenced by the and 2Kidney Research hemodialysis procedure, which stimulates proinflammatory and prothrombotic pathways. Institute, University of Washington, Seattle, Washington; Design, setting, participants, & measurements We compared HDL-associated proteins in 143 participants who 4Department of initiated hemodialysis within the previous year with those of 110 participants with advanced CKD from the Quantitative Health Hemodialysis Fistula Maturation Study. We quantified concentrations of 38 HDL-associated proteins relative to Sciences, Lerner total HDL protein using targeted mass spectrometry assays that included a stable isotope–labeled internal Research Institute, standard. We used linear regression to compare the relative abundances of HDL-associated proteins after Cleveland Clinic, # Cleveland, Ohio; adjustment and required a false discovery rate q value 10% to control for multiple testing. We further assessed 5Department of the association between hemodialysis initiation and cholesterol efflux capacity in a subset of 80 participants. Medicine, Cleveland Clinic Lerner College ResultsAfter adjustment for demographics,comorbidities, and other clinical characteristics, eight HDL-associated of Medicine of Case fi Western Reserve proteins met the prespeci ed false discovery threshold for association. Recent hemodialysis initiation was University, Cleveland, associated with higher HDL-associated concentrations of , A2, and A4; hemoglobin-b; Ohio; 6Department of haptoglobin-related protein; cholesterylester transfer protein; phospholipid transfer protein; and apo E. The trend Medicine, Vanderbilt for participants recently initiating hemodialysis for lower cholesterol efflux capacity compared with individuals University, Nashville, Tennessee; and with advanced CKD did not reach statistical significance. 7 Division of Nephrology and Conclusions Compared with advanced CKD, hemodialysis initiation within the previous year is associated with Hypertension, higher concentrations of eight HDL proteins related to inflammation and metabolism. Identified associations University of Utah differ from those recently observed for nondialysis-requiring CKD. Hemodialysis initiation may further School of Medicine, Salt Lake City, Utah impair cholesterol efflux capacity. Further work is needed to clarify the clinical significance of the identified proteins with respect to cardiovascular risk. Correspondence: Clin J Am Soc Nephrol 13: 1225–1233, 2018. doi: https://doi.org/10.2215/CJN.11321017 Dr. Ke Wang, Kidney Research Institute, Harborview Medical Center, 325 9th Introduction proatherosclerotic processes, such as LDL oxidation, Avenue, Box 359606, Cardiovascular disease is the leading cause of death and macrophage activation, and platelet adhesion (2,6). Seattle, WA 98104. disability among patients on chronic dialysis, account- Among patients on chronic dialysis, absolute HDL Email: kewang@ uw.edu ing for approximately one half of all deaths (1). CKD is cholesterol concentrations are inconsistently associ- associated with a unique pattern of dyslipidemia that may ated with cardiovascular risk, suggesting that func- contribute to cardiovascular risk. Specific lipid abnormal- tional alterations of HDL particles may be clinically ities observed in patients with CKD include an increase in important (7). In this regard, several studies have circulating , a reduction in total HDL choles- shown lower cholesterol efflux capacity and a greater terol, and a disposition towards dense atherogenic LDL abundance of inflammatory proteins in HDL particles particles (2,3). Hepatic hydroxymethyl glutaryl–CoA re- obtained from patients on chronic hemodialysis com- ductase inhibitors, which specifically target LDL choles- pared with healthy individuals (8–12). However, these terol, do not reduce cardiovascular outcomes in clinical findings do not distinguish the potential effects of trials of patients on chronic dialysis (4,5). longstanding kidney failure from those of the hemo- HDL particles are circulating noncovalently bound dialysis procedure itself, which stimulates proinflam- assemblies of and proteins that mediate reverse matory and prothrombotic pathways. A recent study of cholesterol transport by retrieving excess cholesterol HDL composition among patients not on dialysis from peripheral tissues. HDL particles exhibit anti- across a wide range of kidney function showed asso- oxidative and anti-inflammatory functions and inhibit ciations of lower eGFR with higher HDL-associated www.cjasn.org Vol 13 August, 2018 Copyright © 2018 by the American Society of Nephrology 1225 1226 Clinical Journal of the American Society of Nephrology

concentrations of retinol binding protein 4 (RBP4) and APOC3 chromatography-tandem mass spectrometry on a Q-Exactive and lower HDL-associated concentrations of APOL1 and mass spectrometer (Thermo). The same amount of HDL vitronectin (13). protein (10 mg) was analyzed for each sample. We sought to estimate the effect of the hemodialysis procedure on the HDL proteome. To accomplish this goal, Data Reduction we measured 38 HDL-associated proteins in 143 patients The peak area for each endogenous peptide was nor- who recently initiated hemodialysis and 110 patients who malized to the peak area of an internal standard peptide had advanced CKD and were awaiting dialysis initiation in from the stable isotope–labeled internal standard protein to the Hemodialysis Fistula Maturation (HFM) Study. We generate a peak area ratio for each peptide in each sample. hypothesized that dialysis initiation would be associated Peak area ratios for each protein were averaged, and protein with structural transformation of the HDL proteome to a peak area ratios were normalized to protein peak area ratios fl more in ammatory phenotype, which in turn, would lead for calibrator samples included in each digestion batch. to impaired HDL function. Because the same amount of HDL protein was used for each sample, changes in relative protein concentrations represent changes in the amount of protein per 10 mgtotalHDL Materials and Methods protein. We quantified 38 proteins that are abundant in the Study Population HDL proteome and have been previously assayed using the We analyzed plasma samples collected from the HFM same protocol (13,15–17). Study, a prospective cohort study of arteriovenous fistula (AVF) maturation that enrolled 602 participants undergoing planned creation of an upper extremity AVF at seven study Cholesterol Efflux Capacity fl sites across the United States (14). The institutional review Serum HDL cholesterol ef ux capacity was assessed in 3 boards of each of these institutions and the Data Coordinat- J774 macrophages labeled with H-cholesterol and stimulated fl ing Center approved the study. Each participant provided with a cAMP analog (18). Ef ux by the ATP binding cas- informed consent before study enrollment. The HFM Study sette transporter A1 (ABCA1) pathway was measured with participants were either receiving maintenance dialysis or BHK cells expressing mifepristone-inducible human ABCA1 3 fl expected to start dialysis within 3 months of planned AVF that were radiolabeled with H-cholesterol (19). Ef ux of 3 surgery. Exclusion criteria were age ,18 years old, age $80 H-cholesterol was measured after 4 hours of incubation in years old if not receiving maintenance dialysis, or a life medium with or without serum depleted of apoB (2.8% vol/ fl expectancy of ,9 months. For purposes of this ancillary study, vol). The serum HDL cholesterol ef ux capacity is calculated 3 we excluded 349 participants from our analysis (288 without as the amount of H-cholesterol in the media normalized to 3 adequate plasma sample for HDL proteome assays, 59 HFM the total amount of H-cholesterol in the media and the cells. Study participants who had received maintenance dialysis for .1 year, and two participants who were receiving peritoneal Ascertainment of Other Study Data dialysis), leaving a final analytic sample of 253 participants. At the baseline study visit, the HFM Study personnel used We assessed cholesterol efflux capacity in a subset of 80 study patient interviews and medical records to obtain patient de- participants; we matched 40 participants who initiated he- mographics; medical histories; ESRD history, including modal- modialysis to 40 participants with advanced CKD according ity and number of years undergoing dialysis; social habits; and to age within one year, race, and sex. home medication use. Demographics included age, sex, self- reported race and ethnicity, and smoking status. Comorbid HDL Isolation and Quantification of HDL-Associated conditions included a prevalent history of cardiovascular Proteins disease (angina, myocardial infarction, coronary artery bypass, Blood samples were collected at a baseline preoperative or percutaneous coronary revascularization) and diabetes. The study after overnight fasting. On collection, samples were HFM Study personnel obtained the names of all active oral stored at 280°C, maintained at the National Institute of medications at the time of the study visit; measured height, Diabetes and Digestive and Kidney Diseases Biosample weight, and three resting BPs in the nonaccess arm; and Repository, and then shipped to the University of Wash- extracted hemoglobin levels from medical charts. The University ington for measurement of HDL proteins. of Washington Nutrition Obesity Research Center performed The HDL fraction of plasma (d=1.063–1.210 g/ml) was basic chemistries, including creatinine, albumin, C-reactive purified using a two-step density gradient ultracentrifugation protein, and uric acid. Serum total cholesterol, HDL cholesterol, with potassium bromide (KBr). First, all were and were determined using a standard clinical floated using a KBr solution of 1.210 g/ml and transferred to a chemistry analyzer at the University of Washington Kidney new tube. Second, all lipoproteins less dense than HDL were Research Institute (Beckman Coulter DxC, Indianapolis, IN). floated using a KBr solution of 1.063 g/ml. The lipoproteins at the bottom of each sample after this second step were dialyzed Statistical Analyses and frozen at 280°C before use. Internal standard (15N-labeled We tabulated baseline characteristics of the HFM Study APOA1) was added to each sample, and the lipoproteins participants who initiated hemodialysis within the pre- were denatured using Rapigest (Waters), reduced with vious year versus those who were awaiting dialysis. We dithiothreitol, alkylated using iodoacetamide, and digested used linear regression to compare differences in plasma with trypsin. The Rapigest, particulates, and phospho- concentrations of the 38 HDL-associated proteins between lipids were removed with a phospholipid removal plate these two groups. The outcome variables were the log- (Phenomenex) before being analyzed by nanoflow-liquid transformed concentrations of each HDL-associated protein, Clin J Am Soc Nephrol 13: 1225–1233, August, 2018 HDL Composition and Hemodialysis Initiation, Wang et al. 1227

and the independent variable was dialysis status. Exponen- tiated coefficients from this model can be interpreted as ratios Table 1. Baseline participant characteristics by hemodialysis of geometric means of each HDL protein comparing pa- status tients who recently initiated hemodialysis with patients with Awaiting Recent predialysis CKD. We next performed adjusted analyses to Characteristic Dialysis, Hemodialysis,a control for confounding. The first model included demo- n=110 n=143 graphic adjustments for age, race (black versus nonblack), Age, yr 58 (11) 54 (14) and sex. The second model added adjustments for histories Race of diabetes and prevalent cardiovascular disease, body mass White 72 (65) 54 (38) index, smoking status, statin use, and serum total choles- Black 33 (30) 73 (51) terol, HDL cholesterol, and triglycerides. Other 5 (5) 16 (11) – Men 72 (65) 97 (68) To account for multiple comparisons, we used the Benjamini Smoking Hochberg procedure at a false discovery rate (FDR) q-value Current 21 (19) 24 (17) threshold of 10% to declare statistical significance (20–22). Under Former 48 (44) 41 (29) this approach, 10% of those differences in HDL proteins meeting Never 41 (37) 78 (54) the q-value threshold would be expected to be false positives History of diabetes 73 (66) 77 (54) fi Prevalent 58 (53) 56 (39) (23). A protein needed to achieve signi cance under at least cardiovascular one adjustment model to be explored in further analyses. We disease implemented the FDR approach using the GenABEL package Dialysis duration, mo NA 4.6 (3) Body mass index, 32 (7) 29 (8) in R (24). All other statistical analyses were conducted using 2 open source software R version 3.3.0 (https://www.r-project. kg/m Systolic BP, mm Hg 156 (20) 152 (25) org) and STATA 11 (StataCorp, College Station, TX). Statin use 69 (66) 64 (46) Niacin use 2 (2) 1 (1) Fibrate use 7 (7) 3 (2) Results Insulin use 49 (47) 45 (32) eGFR, ml/min per 13 (4) NA Description of the Study Population 1.73 m2 For this ancillary study, we excluded 288 HFM study C-reactive protein,b 3.4 (1.2–10.7) 5.6 (2.9–23.8) participants who had inadequate plasma sample volume for mg/L performing the HDL proteome assays, 59 participants who Uric acid, mg/dl 7.8 (2.0) 5.3 (1.7) had received maintenance dialysis for longer than 1 year, and Albumin, g/dl 3.6 (0.6) 3.5 (0.7) Hemoglobin, g/dl 10.4 (1.6) 10.3 (1.7) two participants who were receiving peritoneal dialysis, c d fi Total cholesterol, 174 (52) 172 (42) leaving a nal analytic sample of 253 participants. Charac- mg/dl teristics of our 253-person study cohort were generally HDL-C, mg/dl 46 (14)c 51 (17)d c d similar to those of the full 602-person HFM Study population Triglycerides, mg/dl 180 (110) 144 (61) (Supplemental Table 1). Compared with participants who were awaiting dialysis, participants who initiated hemodialysis Values are expressed as mean (SD) or number (percentage). NA, not applicable; HDL-C, HDL cholesterol. within the previous year tended to be younger (54614 versus a 6 The Hemodialysis Fistula Maturation Study participants who 58 11 years old); were more likely to be black (51% versus initiated hemodialysis within the previous year. 30%); had lower prevalence rates of diabetes (54% versus 66%) bValues are expressed as median (interquartile range). cValues are missing for eight participants. and cardiovascular disease (39% versus 53%); and had lower d serum triglycerides and higher serum HDL cholesterol and Values are missing for three participants. C-reactive protein concentrations (Table 1).

Associations of Hemodialysis with HDL-Associated Proteins After adjustment for age, race, and sex (model 1), seven of that account for approximately 90% of total HDL protein 38 HDL-associated proteins met the specified FDR threshold mass (25). for statistical significance (Figure 1). Relative concentrations of significant HDL proteins were higher among participants Associations of Hemodialysis Duration with HDL-Associated recently initiating hemodialysis compared with those await- Proteins ing dialysis. Specifically, recent hemodialysis initiation was In additional analyses restricted to the 143 participants associated with higher concentrations of serum amyloid A2 who initiated hemodialysis within the previous year, the (SAA2), hemoglobin-b, SAA1, haptoglobin-related protein duration of dialysis was not associated with differences in (HPR), cholesterylester transfer protein (CETP), phospho- any of the candidate HDL proteins (Table 3). lipid transfer protein (PLTP), and APOE. The sizes of these associations after model 1 adjustment ranged from 29% Association of Hemodialysis Initiation with Cholesterol greater concentrations of APOE to 128% greater concentra- Efflux Capacity tions of SAA2 (Table 2). After further covariate adjustments Individuals recently initiated on hemodialysis showed under model 2, the association of dialysis initiation with PLTP modestly lower total efflux capacity (11.4% versus 11.8%) was no longer statistically significant, whereas the associa- and ABCA1-mediated efflux capacity (13.6% versus 14.3%) tion with SAA4 reached statistical significance. There were compared with those with advanced CKD; however, this no detectable differences in the relative concentrations of difference was not statistically significant. Adjustment for APOA1 and APOA2, the two most abundant HDL proteins participant demographics and serum HDL cholesterol level 1228 Clinical Journal of the American Society of Nephrology

Figure 1. | Ratios of seven HDL-associated proteins are higher in recent hemodialysis compared to late-stage CKD. Forest plot of ratios of HDL- associated protein concentrations comparing participants on hemodialysis for ,1 year with participants with late-stage CKD. Exponentiated b-coefficients are plotted along with 95% confidence intervals, with q values, calculated after model 1 adjustment for age, race (black versus nonblack), and sex. The x axis is presented in the logarithmic scale. *Significant HDL-associated proteins after performing false discovery rate correction (q#10%). ALB, albumin; A1AT, a1-antitrypsin; CETP,cholesteryl ester transfer protein; CLU, clusterin; CO3, complement C3; CO4, complement C4; HBB, hemoglobin-b; HPR, haptoglobin-related protein; HPT,haptoglobin; IHH, Indian hedgehog protein; KAIN, kallistatin; LCAT, phosphatidylcholine-sterol acyltransferase; LPA, (a); PAFA, platelet-activating factor acetylhydrolase; PCYOX1, prenylcysteine oxidase 1; PLTP,phospholipid transfer protein; PON1, paraoxonase 1; PON3, paraoxonase 3; RBP4, retinol binding protein 4; SAA1, serum amyloid A1; SAA2, serum amyloid A2; SAA4, serum amyloid A4; VDBG, vitamin D binding globulin; VTN, vitronectin.

strengthened the association between dialysis initiation was observed between relative abundance of SAA1- and and impaired cholesterol efflux capacity (Table 4). Total ABCA1-mediated efflux; however, this was not statisti- efflux capacity was highly correlated with ABCA1-mediated cally significant (r=20.14; P value =0.21) (Supplemental efflux capacity (r=0.75). A very weak negative correlation Table 2). Clin J Am Soc Nephrol 13: 1225–1233, August, 2018 HDL Composition and Hemodialysis Initiation, Wang et al. 1229

Table 2. Association of hemodialysis initiation with HDL-associated proteins

Ratio of Protein Concentrations Comparing Protein and Model 95% Confidence Interval q Value Hemodialysis with Late-Stage CKDa

Serum Amyloid A2 Unadjusted 2.21 1.19 to 4.09 Model 1b 2.28 1.19 to 4.35 0.08 Model 2c 3.21 1.60 to 6.43 0.04 Serum Amyloid A1 Unadjusted 1.62 1.07 to 2.45 Model 1 1.69 1.10 to 2.60 0.09 Model 2 1.99 1.26 to 3.16 0.04 Hemoglobin-b Unadjusted 1.89 1.25 to 2.87 Model 1 1.74 1.13 to 2.68 0.08 Model 2 1.92 1.20 to 3.07 0.05 Haptoglobin-Related Protein Unadjusted 1.89 1.42 to 2.52 Model 1 1.45 1.11 to 1.89 0.07 Model 2 1.44 1.09 to 1.89 0.06 Cholesterol Ester Transfer Protein Unadjusted 1.36 1.11 to 1.65 Model 1 1.34 1.09 to 1.65 0.07 Model 2 1.30 1.05 to 1.62 0.08 Phospholipid Transfer Proteind Unadjusted 1.34 1.10 to 1.63 Model 1 1.32 1.08 to 1.61 0.07 Model 2 1.20 0.98 to 1.47 0.28 E Unadjusted 1.34 1.14 to 1.57 Model 1 1.29 1.09 to 1.53 0.07 Model 2 1.31 1.09 to 1.56 0.04 Serum Amyloid A4e Unadjusted 1.17 1.02 to 1.35 Model 1 1.17 1.01 to 1.35 0.14 Model 2 1.25 1.07 to 1.46 0.04

aRatio of geometric means. bModel 1 was adjusted for age, race (black versus nonblack), and sex. cModel 2 adds adjustment for diabetes, body mass index, prevalent cardiovascular disease, smoking, statin use, and serum levels of total cholesterol, HDL-C, and triglycerides. dPLTP was no longer statistically significant after model 2 adjustment. eSAA4 was significant after model 2 adjustment only.

Discussion Noneoftheseproteinswereassociatedwithhemodialysis We observed associations of recent hemodialysis initiation initiation in our study, and conversely, none of the proteins with a greater relative abundance of eight HDL-associated that we identified were associated with reduced GFR among proteins. The identified HDL constituents in this study are patients in the CKD cohort study. These findings suggest that markers or mediators of inflammatory, atherosclerotic, and kidney disease and hemodialysis may each uniquely affect the lipid metabolism pathways. None of these HDL-associated HDL proteome. proteins were associated with lower eGFR in a recent study Previous studies have reported differences in HDL- of patients with nondialysis-requiring CKD across a wide associated proteins when comparing patients on chronic range of kidney function (13). We also observed a trend hemodialysis with healthy controls. Holzer et al. (8) found toward lower HDL cholesterol efflux capacity in a subset of lower relative concentrations of APOA1 and APOA2 and study participants. Although the possibility of confounding higher concentrations of SAA1, albumin, and APOC3 in 27 cannot be excluded, these findings suggest a possible effect of patients on hemodialysis compared with 19 healthy controls. the hemodialysis procedure on the composition and function Subsequent studies have reported associations of hemodial- of the HDL proteome. ysis with lower HDL concentrations of APOA1 and APOA2 The observed associations of recent dialysis initiation with (9,11,12) and higher concentrations of SAA (9,11,12) and HDL-associated proteins in this study differ from those re- APOC3 (10–12). Previous studies have also reported associ- cently reported for reduced eGFR in patients with non- ations of chronic hemodialysis with higher HDL concentra- dialysis CKD. Among 509 patients with CKD not receiving tions of APOC2 (9–11), lipoprotein-associated phospholipase dialysis, each 15-ml/min per 1.73 m2 lower eGFR was as- A2 (8), surfactant protein B (9), a1-microglobulin/bikunin sociated with higher HDL concentrations of RBP4 and APOC3 precursor (9,12), a1-acid glycoprotein 2, RBP4, a1-antitrypsin, and lower HDL concentrations of APOL1 and vitronectin (13). APOA4, HPR, and transthyretin (12). Most of these 1230 Clinical Journal of the American Society of Nephrology

Table 3. Association of dialysis duration with HDL-associated proteins

Ratio of Protein Concentrations per Protein and Model 95% Confidence Interval P Value Month Longer on Chronic Dialysisa

Serum Amyloid A2 Unadjusted 1.01 0.89 to 1.13 Model 2b 1.03 0.91 to 1.16 0.61 Serum Amyloid A1 Unadjusted 1.05 0.97 to 1.14 Model 2 1.05 0.97 to 1.15 0.19 Hemoglobin-b Unadjusted 0.98 0.90 to 1.06 Model 2 0.97 0.90 to 1.06 0.61 Haptoglobin-Related Protein Unadjusted 1.02 0.97 to 1.07 Model 2 1.00 0.96 to 1.06 0.75 Cholesterol Ester Transfer Protein Unadjusted 0.99 0.96 to 1.03 Model 2 0.99 0.96 to 1.03 0.79 Phospholipid Transfer Protein Unadjusted 1.01 0.97 to 1.04 Model 2 1.00 0.97 to 1.04 0.84 Unadjusted 1.00 0.97 to 1.03 Model 2 1.00 0.97 to 1.03 1.03 Serum Amyloid A4 Unadjusted 1.00 0.98 to 1.03 Model 2 1.00 0.98 to 1.03 0.53

aRatio of geometric means. bModel 2 was adjusted for age, race (black versus nonblack), sex, diabetes, body mass index, prevalent cardiovascular disease, smoking, statin use, and serum concentrations of total cholesterol, HDL cholesterol, and triglycerides.

identified HDL proteins are acute-phase reactants and compared with individuals without kidney disease (8,11,30). A important mediators of immune response and thrombosis recent study showed impaired HDL-mediated cholesterol pathways (12,26). Previous studies of chronic dialysis are efflux across varying stages of CKD compared with healthy limited by small sample size and comparison of patients on controls (31). Therefore, our results suggest that hemodial- dialysis with healthy controls, which conflates potential ysis may be independently associated with impaired HDL- effects of kidney disease with those of the hemodialysis mediated cholesterol efflux. procedure. Hemodialysis may independently provoke a unique We observed that hemodialysis initiation was associated inflammatory microenvironment that could promote ath- with a strong trend toward impaired cholesterol efflux erogenic transformation of the HDL proteome and impair capacity from lipid-laden macrophages. Total cholesterol HDL function. We observed higher serum C-reactive efflux is mediated through several known pathways, including protein concentrations among study participants who ABCA1, ABCG1, scavenger receptor B1, and aqueous diffu- recently initiated hemodialysis compared with those with sion (27). Both animal and human studies have shown that late-stage CKD. Prior studies have shown that hemodialysis ABCA1-mediated efflux is the key pathway for HDL reverse is associated with increased inflammation independent of cholesterol transport (18,28). In the general population, lower kidney function. Patients on hemodialysis exhibit higher cholesterol efflux capacity is associated with higher risks of serum levels of IL-6 compared with patients who are uremic cardiovascular events and death (29). Several studies have and are not on dialysis and higher HDL concentrations of shown lower cholesterol efflux among patients on dialysis inflammatory proteins compared with individuals on

Table 4. Association between dialysis initiation and cholesterol efflux capacity

Unadjusted Adjusted Cholesterol Advanced Hemodialysis Adjusted Difference Difference Efflux Capacity CKD (%)a (%)a P Value (%; 95% CI) (%; 95% CI)b

Total 11.8 (2.7) 11.4 (2.4) 20.4 (21.5 to 0.8) 20.9 (22.0 to 0.1) 0.07 ABCA1 14.3 (3.1) 13.6 (2.2) 20.6 (21.8 to 0.6) 21.0 (22.2 to 0.1) 0.08

95% CI, 95% confidence interval; ABCA1, ATP binding cassette transporter A1. an=40. bAdjusted for age, race (black versus nonblack), sex, and HDL-C cholesterol level. Clin J Am Soc Nephrol 13: 1225–1233, August, 2018 HDL Composition and Hemodialysis Initiation, Wang et al. 1231

peritoneal dialysis (11,32). Serial monitoring of inflammatory Identified proteins reside in pathways of inflammation, lipid markers after dialysis initiation shows that hemodialysis does metabolism, and and differ from those pre- not attenuate the proinflammatory and pro-oxidative milieu viously associated with reduced kidney function in patients that accompanies kidney disease (33). In fact, hemodial- with CKD. Our findings suggest that the hemodialysis ysis may further exacerbate the existing inflammatory procedure may provoke a unique pattern of dysfunctional milieu of uremia via several plausible mechanisms, such HDL particles that are less prevalent in late-stage CKD. as activation of the immune response through blood in- Further work is needed to confirm these suspected changes teraction with dialysis membrane and back filtration of and clarify their clinical significance. endotoxins contained within the dialysate (34,35). HDL proteins associated with hemodialysis in this study are Acknowledgments known to be involved in inflammatory, lipid metabolism, and The authors would like to acknowledge the technical assistance thrombosis pathways. SAA is an activator of the innate immune of Jennifer Wallace in the preparation of the HDL particles. system and can stimulate thrombosis (36,37). Higher HDL- The Hemodialysis Fistula Maturation Study was funded by associated SAA concentrations in healthy individuals and grants U01DK082218, U01DK082222, U01DK082232, U01DK082236, patients on hemodialysis correlate with lower HDL cholesterol U01DK082240, U01DK082179, U01DK082189 from the National efflux capability (8) and are associated with cardiovascular Institute of Diabetes and Digestive and Kidney Diseases. This study mortality (38). CETP and PLTP mediate HDL cholesterol was funded by University of Washington Nutrition Obesity Research transport. Although CETP is thought to be proatherogenic in Center grants P30DK035816, T32DK00746733 (to K.W.), R01HL111375 individuals with coronary artery disease (39), evidence (to A.N.H.), T32HL007028 (to C.M.H.), and R01DK094891 (to B.K.). suggests that this protein may protect against thrombosis in Because I.H.d.B. is a Deputy Editor of the Clinical Journal of the patients on hemodialysis (40,41). Increased PLTP activity is American Society of Nephrology, he was not involved in the peer re- associated with accelerated atherosclerosis in mice and view process for this manuscript. Another editor oversaw the peer cardiovascular events in the Framingham Heart Study review and decision-making process for this manuscript. Rajnish (42,43). HDL enrichment of APOE can interfere with HDL Mehrotra, the Editor-in-Chief, is at the same institution as some of the authors, including the Deputy Editor, and therefore, was also not metabolism and instigate atherogenesis (44,45). The HDL3 fraction isolated from patients with coronary artery disease is involved in the peer review process for this manuscript. enriched with APOE, and higher APOE levels are associated with cardiovascular mortality (46,47). HPR is an acute-phase Disclosures fi reactant with a high binding affinity for hemoglobin in the B.K. hasreceivedconsulting feesfrom Sano Inc. The laboratoryof setting of hemolysis. HDL abundances of both HPR and A.N.H. receives grant funding from Waters, a mass spectrometry hemoglobin are increased in animal models of atherosclerosis manufacturer. The other authors indicate no disclosures. and human subjects with coronary heart disease (48,49). One strength of our study is the comparison of the HDL References proteome in patients on hemodialysis with that of patients 1. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY: Chronic with advanced CKD awaiting dialysis from within the same kidney disease and the risks of death, cardiovascular events, and parent study. We used validated, targeted mass spectrometry hospitalization. NEnglJMed351: 1296–1305, 2004 2. Vaziri ND: Dyslipidemia of chronic renal failure: The nature, assays with internal standards to quantify the protein content mechanisms, and potential consequences. Am J Physiol Renal of isolated HDL particles, and we adjusted for differences in Physiol 290: F262–F272, 2006 patient characteristics that were measured using uniform 3. de Boer IH, Astor BC, Kramer H, Palmas W, Seliger SL, Shlipak data collection procedures in the HFM Study. MG, Siscovick DS, Tsai MY, Kestenbaum B: Lipoprotein abnor- The most important limitation of the study is the possibility malities associatedwith mild impairment of kidney function in the multi-ethnic study of atherosclerosis. Clin J Am Soc Nephrol 3: of residual confounding, in which unmeasured differences in 125–132, 2008 the characteristics of the patients not on dialysis versus 4. Wanner C, Krane V, Ma¨rz W, Olschewski M, Mann JF, Ruf G, patients on dialysis in this study may have distorted the Ritz E; German Diabetes and Dialysis Study Investigators: observed associations. For example, patients initiating Atorvastatin in patients with type 2 diabetes mellitus undergoing hemodialysis. N Engl J Med 353: 238–248, 2005 chronic dialysis may have had more severe uremia, which 5. Fellstro¨m BC, Jardine AG, Schmieder RE, Holdaas H, Bannister K, can lead to transformation of the HDL proteome independent Beutler J, Chae DW, Chevaile A, Cobbe SM, Gro¨nhagen-Riska C, of the dialysis procedure. 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Title: Alteration of high-density lipoprotein protein composition with hemodialysis initiation

Supplemental Material

Supplemental Table 1: Baseline participant characteristics of HFM study compared to HFM proteome study

Supplemental Table 2: Correlation between relative HDL protein abundance and ABCA1-mediated cholesterol efflux

1

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Supplemental Table 1: Baseline participant characteristics of HFM study compared to HFM proteome study 1HFM proteome HFM n=253 n=602 Age (years) 55.9 (12.9) 55.1 (13.4) Race White 126 (49.8) 283 (47.0) Black 106 (41.9) 264 (43.8) Other 21 (8.30) 55 (9.1) Male sex 169 (66.8) 423 (70.3) Smoking Current 45 (17.8) 106 (17.6) Former 89 (35.2) 222 (36.9) Never 119 (47.0) 274 (45.5) History of diabetes 150 (59.3) 353 (58.6) Prevalent cardiovascular disease 114 (45.1) 290 (48.2) Maintainence hemodialysis 143 (56.5) 383 (63.6) 2Dialysis duration (months) 4.6 (3.4) 4.3 (3.2) BMI (kg/m2) 30.3 (7.7) 30.4 (7.5) Systolic blood pressure (mmHg) 153.5 (23.3) 151.2 (23.8) Statin use 133 (54.5) 336 (55.8) Niacin use 3 (1.2) 10 (1.7) Fibrate use 10 (4.1) 22 (3.6) Insulin use 94 (38.5) 232 (38.5) 3Estimated GFR (mL/min/1.73m2) 12.8 (4.4) 13.7 (4.7) Albumin (g/dL) 3.5 (0.6) 3.6 (0.6) Hemoglobin (g/dL) 10.3 (1.6) 10.5 (1.7) GFR=glomerular filtration rate

1Excludes HFM Study participants on hemodialysis for longer than one year, receiving peritoneal dialysis, or whose plasma samples had insufficient volume for HDL proteome analysis. 2Excludes participants on hemodialysis for longer than one year. 3Excludes participants on dialysis.

2

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Supplemental Table 2: Correlation between HDL protein and ABCA1 efflux

HDL-protein1 Correlation coefficient2 p-value

SAA2 -0.06 0.62

SAA1 -0.14 0.21

HBB -0.05 0.65

HPR 0.10 0.36

CETP 0.06 0.61

PLTP 0.05 0.68

APOE 0.03 0.77

SAA4 0.02 0.87

1 Relative HDL protein abundance per 10µg total HDL particle

2 Participants with measured cholesterol efflux capacity (n=80)

3