CKD Increases Carbonylation of HDL and Is Associated with Impaired Antiaggregant Properties

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Nans Florens ,1,2 Catherine Calzada,1 Sandrine Lemoine,1,2 Marie Michèle Boulet,1 Nicolas Guillot ,1 Christophe Barba,1 Julie Roux,1 Fréderic Delolme,3 Adeline Page,3 Jean Michel Poux,4 Maurice Laville,4 Philippe Moulin,1,5 Laurent Soulère,6 Fitsum Guebre-Egziabher,1,2 Laurent Juillard,1,2 and Christophe O. Soulage1

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Background CKD is associated with increased oxidative stress that correlates with occurrence of cardiovascular events. Modifications induced by increased oxidative stress particularly affect circulating lipoproteins such as HDL that exhibit antiatheromatous and antithrombotic properties in vitro. Methods To explore the specific role of oxidative modifications of HDL in CKD and their effect on the platelet-targeting antiaggregant properties of HDL, we used a CKD (5/6 nephrectomy) rabbit model. For ex vivo assessment of the antiaggregant properties of HDL, we collected blood samples from 15 healthy volunteers, 25 patients on hemodialysis, and 20 on peritoneal dialysis. We analyzed malondialdehyde, 4-hydroxynonenal (HNE), and 4-hydroxy-2-hexenal protein adduct levels. Platelet aggregation and activation were assessed by aggregometry, thromboxane B2 assay, or FACS. We modified HDL from controls by incubating it overnight at 37°C with 100 mMofHNE. Results HDL from CKD rabbits and patients on hemodialysis had HNE adducts. The percentage of platelet aggregation or activation induced by collagen was significantly higher when platelets were incubated with HDL from CKD rabbit and hemodialysis groups than with HDL from the control group. In both rabbits and humans, platelet aggregation and activation were significantly higher in the presence of HNE-modified HDL than with HDL from their respective controls. Incubation of platelets with a blocking antibody directed against CD36 or with a pharmacologic inhibitor of SRC kinases restored the antiaggregative phenotype in the presence of HDL from CKD rabbits, patients on hemodialysis and peritoneal dialysis, and HNE-modified HDL. Conclusions HDL from CKD rabbits and patients on hemodialysis exhibited an impaired ability to inhibit platelet aggregation, suggesting that altered HDL properties may contribute to the increased cardiovascular risk in this population.

JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019111205

CKD is recognized as a major cardiovascular risk induced by increased oxidative stress particularly affect factor.1–3 It is involved in the onset of cardiovascu- circulating lipoproteins such as HDL that exhibit anti- lar events such as myocardial infarction, peripheral atheromatous and antithrombotic properties in vitro. arterial disease, and cerebral ischemia.1 Cardiovas- cular mortality remains the major cause of death in patients on hemodialysis (HD) and peritoneal di- Received November 23, 2019. Accepted March 22, 2020. alysis (PD) despite the constant improvement of Published online ahead of print. Publication date available at RRT.4 CKD is associated with increased oxidative stress www.jasn.org. that is correlated with the occurrence of cardiovascular Correspondence: Dr. Nans Florens, CarMeN Laboratory, UMR IN- – events.5 7 This stress is associated with the accumu- SERM U.1060, INSA-Lyon, Building IMBL, 15 avenue Jean Capelle, lation of many uremic toxins,8 some of which have 69621 Villeurbanne cedex, France. Email: nans.ﬂ[email protected] – recognized cardiovascular effects.9 12 Modiﬁcations Copyright © 2020 by the American Society of Nephrology

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There are many mechanisms that lead to lipoprotein mod- Significance Statement ification, both enzymatic and nonenzymatic, and these affect different lipoprotein sites.13 Oxidized and carbamylated LDL CKD is associated with increased oxidative stress that correlates are present at higher concentrations in patients with CKD with the occurrence of cardiovascular events. Oxidative stress in- fi compared with patients who are healthy5,14 and play a major duces modi cations that particularly affect circulating lipoproteins such as HDL that exhibit atheroprotective properties in vitro. role in the onset and aggravation of atherosclerotic le- However, information about the antithrombotic properties of HDL sions.14,15 The concentration of oxidized and carbamylated in CKD is lacking. The authors demonstrate that HDL from a CKD LDL are, however, lowered by statins.16–18 HDL is considered rabbit model and patients on hemodialysis exhibited an impaired to be antiatherogenic as a result of its antiaggregant, anti- ability to inhibit platelet aggregation, suggesting that properties of inflammatory, and antiapoptotic properties,19 as well as its altered HDL may contribute to the increased cardiovascular risk in this patient population. They also describe the putative role of fl 20 ability to induce cholesterol ef ux from macrophages, a carbonylation by 4-hydroxynonenal adduction in these properties. mechanism known to be atheroprotective.21 HDL is not af- This study provides important insights into the potential implication fected by conventional hypolipidemic strategies, and func- of HDL modifications in atherothrombosis and cardiovascular tional studies in CKD have found impaired biologic effects, morbidity and mortality among patients on dialysis. including decreased capacity of macrophage cholesterol fl 22–24 24 ef ux and antioxidation, as well as impaired endothelial months. Exclusion criteria were the presence of diabetes mel- 25 fi protection. These disorders become more signi cant with litus, ongoing inflammatory disease, liver cirrhosis, recent car- 25 CKD progression. In parallel, HDL oxidation has been re- diovascular event (,3 months; myocardial infarction, stroke, lated to the onset of cardiovascular events in patients with acute peripheral artery occlusion), uncontrolled anemia, coa- 26 fi CKD. These functional modi cations could partly explain gulopathy, and body mass index .35 kg/m2. The study was the failure of statins to reduce the cardiovascular risks associ- conducted in accordance with the Declaration of Helsinki and 27,28 ated with HD. was approved by the institutional review board (Comité Platelets are the target of antiaggregant treatments that have de protection des personnes Lyon Sud Est IV, Centre Léon fi been shown to be bene cial in reducing cardiovascular events Berard, reference, L16-57). A written informed consent was in at-risk populations. Oxidized LDL of patients who are obese obtained from all subjects. Blood samples were obtained by 29,30 and diabetic has shown some proaggregating properties. venipuncture on EDTA-coated tubes. Blood samples were In CKD, carbamylated LDL exhibits proaggregant properties centrifuged at 3500 3 g for 10 minutes to isolate plasma and 31 through binding with lectin-like oxidized LDL receptor 1, were then stored at 280°C until use (Supplemental Methods). and oxidized LDL through binding to the CD36 receptor.32 Oxidation of HDL leads to a loss of antiaggregation properties in some populations,33 although this observation was not Animal Procedures found in type 2 diabetes,34 where oxidized HDL has shown All experiments were performed under the authorization antiaggregant properties through a Scavenger receptor class B number 69-266-0501 and agreed with the guidelines laid type 1 (SR-B1)–mediated pathway.35 However, to the best of down by the French Ministry of Agriculture (number 2013- our knowledge, there is no published data regarding the effect 118) and the European Union Council Directive for the pro- of CKD on the antiaggregant properties of HDL. tection of animals used for scientific purposes of September In this study, we aimed to explore the specific role of CKD 22, 2010 (2010/63UE). Adult male white New Zealand rabbits in the oxidation profile of HDL and the effects of HDL mod- (CEGAVssc, Saint Mars d’Egrenne, France) were housed in ification on platelet aggregation in a rabbit model of CKD. We individual cages at constant ambient temperature (21–23°C) then aimed to assess, ex vivo, the antiaggregant properties of and humidity (45%–50%) with a 12-hour light cycle. All an- HDL isolated from patients on HD and PD. imals had free access to tap water. After a 7-day period of acclimation, rabbits were randomized to either the 5/6 nephrectomy group or the control group. Nephrectomy was per- METHODS formed as described by Gotloib et al.36 Briefly, rabbits were anesthetized using an intramuscular injection of ketamine Subjects and Ethics Statement (50 mg/kg), xylazine (5 mg/kg), and acepromazine Patients were sampled at the Lyon teaching hospitals. Control (0.5 mg/kg; Centravet, Lapalisse, France). The rabbit was patients were healthy volunteers for a living kidney donation, placed in the right lateral decubitus position. Local anesthesia hospitalized for their predonation laboratory and clinical was performed by a subcutaneous injection of xylocaine 2% workup. Patients on HD were sampled within the HD unit (2 ml) at the incision site. The left kidney was externalized and of the Edouard Herriot Hospital (Lyon, France) before the the perirenal adipose tissue was gently dissected. The two poles midweek session. Patients on PD were sampled in the PD of the kidney were electrocoagulated using an electric needle unit of the Association pour l’Utilisation du Rein Artificiel to produce a two-third reduction of the left kidney mass. At (AURAL; Lyon, France). Inclusion criteria were participants 1 week after surgery, a unilateral right nephrectomy was per- aged $18 years who were undergoing HD or PD for .6 formed. The kidney pedicle was carefully sectioned between

2 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH the kidney and the ligature. The control animals underwent 6 hours 30 minutes at 4°C after an adjustment of plasma den- the same procedures (general anesthesia, skin, and muscle sity to 1.21 g/ml with KBr. incisions) followed by a simple kidney mobilization. All rab- For platelet aggregation and the copper-induced HDL ox- bits were given buprenorphine (0.05 mg/kg subcutaneously, idation assay, freshly isolated HDL samples were used to pre- two times a day) for 2 days to prevent postsurgical pain. Food vent HDL ultrastructure modification due to freezing.40 After intake and body weight were measured on a daily and twice- ultracentrifugation, lipoproteins were extensively dialyzed weekly basis, respectively (Supplemental Methods). against PBS with 1 mM EDTA for 3 hours, twice at room temperature and then overnight at 4°C. A last dialysis without Measurement of GFR EDTA was performed just before the platelet aggregation and The GFR in rabbits was measured through the kinetics of de- copper-induced oxidation. HDL samples were stored at 4°C crease of plasma concentration of iohexol as described in Flo- for a maximum of 48 hours before the experiments. rens et al.37 Rabbits were anesthetized with an intravenous injection of 27.5 mg/kg of sodium pentobarbital (Centravet). Biochemistry A PE-50 catheter was introduced in the left jugular vein for the Serum creatinine measurement was performed using the Sie- injections and a PE-60 catheter was inserted in the left carotid mens enzymatic method (on the Dimension Vista System; artery for the blood sampling. A 1-ml bolus of iohexol (Om- Siemens Healthcare, Erlangen, Germany). Urea was measured nipaque 300; GE Healthcare, Chicago, IL) was performed and using the urease test (Vista 1500). Cholesterol and triacylgly- the timer was started after flushing the residue left in the jug- cerol levels were measured using enzymatic kits (Biomerieux, ular catheter with a saline solution. Blood was sampled at 5, Marcy l’Etoile, France). HDL concentration was measured 15, 30, 45, 60, 120, and 180 minutes on lithium- with an enzymatic kit (Abcam, Paris, France). Malondialde- heparin–coated tubes. The plasma was aliquoted after centri- hyde (MDA) was measured by HPLC coupled to ultraviolet- fugation at 5900 3 g for 2 minutes and stored at 220°C. The visible detection (Diode Array Detector) as described by serum iohexol concentration was measured by HPLC as pre- Grotto et al.41 Antioxidant activity (AOA) of the plasma was viously described38 and GFR was calculated using a bicom- measured as described by Koracevic et al.42 Proteinuria was partmental model equation. measured using the Bradford protein assay (BioRad, Marne-la- Coquette, France) using BSA as a standard. Euthanasia, Necropsy, and Renal Histology At the end of the GFR measurement, rabbits were deeply an- Lipoprotein Assays esthetized with an overdose of sodium pentobarbital MDA concentration in HDLwas determined by HPLC accord- (70 mg/kg, intravenously). Blood was removed by cardiac ing to the method described by Therasse and Lemonnier.43 puncture and placed in EDTA-coated tubes. After a centrifu- Anti–4-hydroxynonenal (HNE)-Michael adduct (reference gation at 1250 3 g for 10 minutes, plasma was aliquoted and 393207) and anti–4-hydroxy-2-hexenal (HHE)-Michael ad- stored at 280°C. Urine was obtained via a direct bladder duct (reference NOF-N213730-EX) antibodies were obtained puncture and stored at 220°C. Kidneys were removed, from Calbiochem (San Diego, CA) and Cosmobio (Tokyo, weighed, and stored in buffered formalin 10% (w/v) for his- Japan), respectively. A total of 50 mg HDL was loaded directly tologic examination. Kidneys were dehydrated using ethanol, onto a nitrocellulose membrane using the Bio-Dot apparatus embedded in paraffin, and sliced. Hematoxylin Erythrosine (BioRad). Following saturation with 5% BSA, membranes were Saffron and Sirius Red stainings were performed. probed overnight with primary antibodies: anti-HHE-Michael adducts or anti-HNE-Michael adducts. After incubation with Isolation of Lipoproteins from the Plasma horseradish peroxidase–coupled secondary antibodies, mem- Lipoproteins were separated from plasma by stepwise potas- branes were processed for chemiluminescence (ECL Plus; GE sium bromide (KBr) density gradient ultracentrifugation as Healthcare) and quantitated by densitometry using ImageJ described by Havel et al.39 Briefly, plasma was fractionated in software (National Institutes of Health, Bethesda, MD). the Beckman ultracentrifuge with a TLA 100.3 Rotor (Beck- 8-Isoprostane was measured using an immunoassay (Bertin man, Brea, CA). A first centrifugation at 100,000 3 g for Pharma, Montigny Le Bretonneux, France). HNE was synthe- 3 hours 30 minutes at 15°C was performed to remove the tized as described by Soulère et al.44 and was diluted in DMSO. top layer corresponding to VLDL and chylomicrons. Then, HNE was added to HDL solutions to final concentrations of 1, the plasma density was adjusted to 1.063 g/ml with KBr 10, 50, or 100 mM. After a 16 hour (overnight) incubation at (M5119.01 g/mol). After a second centrifugation at 100,000 37°C in a water bath, HDL samples were dialyzed three times 3 g for 5 hours at 4°C, the orange ring, corresponding to LDL, against PBS to remove the free fraction of HNE. was collected. Finally, plasma density was adjusted to 1.21 g/ml with KBr and, after centrifugation at 100,000 3 g for 6 hours Platelet Aggregation, Activation, and Intracellular 30 minutes at 4°C, the orange ring corresponding to HDL was Pathway Assays collected. For the isolation of all of the lipoproteins together, a Blood was collected at the regional blood center from healthy single ultracentrifugation was performed at 100,000 3 g for volunteers who had not ingested any aspirin or any other

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Table 1. General characteristics of sham-operated (control) and 5/6 Arbor, MI). After preincubation with nephrectomized rabbits HDL and antibodies or pharmacologic Characteristics Control (N59) CKD (N58) P Value inhibitor, platelets were activated with fi Biometry collagen and then xed in 5% parafor- Body weight (kg) 3.5 (3.2–3.9) 3.1 (3.0–3.3) 0.04 maldehyde and stained with anti-CD62/ Body weight gain (kg) 1.0 (0.9–1.2) 0.7 (0.6–0.8) 0.001 P-selectin (Thermo Fisher Scientific, Kidney weight (g) 19 (16–22) 14 (12–16) 0.008 San Jose, CA) according to the manufac- Kidney weight (g/kg body wt) 5 (4–7) 5 (4–5) 0.21 turer’sprotocolandanalyzedbyflow Biologic features cytometry.47 Urea (mmol/L) 6.8 (5.6–8.4) 12.8 (12.6–19.2) ,0.001 m – – , Creatinine ( mol/L) 57 (52 75) 193 (154 270) 0.001 Copper-Induced HDL Oxidation – – Bicarbonate (mmol/L) 20.1 (16.8 24.0) 21.0 (14.6 22.7) 0.70 Aliquots of freshly dialyzed HDL (50 mg Glucose (mmol/L) 5.6 (4.8–6.9) 7.4 (7.1–8.1) 0.04 – – , proteins) were oxidized in the presence Total cholesterol (mg/dl) 47 (37 68) 109 (73 198) 0.001 m HDL cholesterol (mg/dl) 29 (18–37) 56 (35–85) 0.04 of 2.5 Mofcoppersulphatewithina HDL-C/TC ratio 0.66 (0.33–0.85) 0.49 (0.35–0.53) 0.27 day after the last dialysis. The oxidation Triacylglycerols (mg/dl) 270 (228–677) 316 (165–719) 0.84 was monitored by measuring the forma- Protidemia (g/L) 59 (48–69) 39 (32–49) 0.02 tion of conjugated dienes at 234 nm.48 Proteinuria (g/mmol creatinine) 617 (60–1287) 19 (9–56) 0.20 Iohexol clearance Mass Spectrometry GFR (ml/min per kg) 4.3 (4.1–4.8) 2.2 (1.8–2.5) 0.008 Samples were reduced, alkylated, and di- Oxidative stress markers (plasma) gested with trypsin at 37°C overnight. m – – MDA ( mol/L) 0.8 (0.5 1.2) 2.3 (1.2 3.0) 0.009 They were desalted with spin column – – AOA (mmol/L) 1.0 (0.8 1.1) 0.8 (0.7 0.9) 0.02 C18, dried, and then analyzed in tripli- Data are expressed as median (interquartile range). P,0.05 was considered as significant by Mann– Whitney test. Creatinine, 30.011 for mg/dl; urea, 32.8 for mg/dl. HDL-C, HDL cholesterol; TC, total cate using an UltiMate 3000 RSLCnano cholesterol. (Thermo Fisher Scientific) coupled online with a Q-Orbitrap (Q Exactive HF; Thermo Fisher Scientific). Briefly, pep- nonsteroidal anti-inflammatory drug in the previous 10 days. tides were separated on a C18 nanocolumn with a linear gra- Platelets were prepared for the assays as described by Lagarde dient of acetonitrile and analyzed in a Top 20 HCD (higher et al.45 The platelet function test was carried out according to collision dissociation) data-dependent mass spectrometer. the Born turbidimetric method.46 Platelet aggregation was Data were processed by database searching using SequestHT measured in isolated platelets in a dual-channel aggregometer (Thermo Fisher Scientific) with Proteome Discoverer 2.2 soft- (Chrono-log; Coulter, Margency, France). Platelet suspen- ware (Thermo Fisher Scientific) against a human Swissprot sions were preincubated for 5 minutes at 37°C in the presence database and quantified with a label-free quantitation ap- or absence of lipoproteins (0.025 mg of proteins/ml for rabbit, proach. Precursor and fragment mass tolerance were set at 0.050 mg/ml for human) and stimulated with threshold con- 10 ppm and 0.02 Da, respectively. Trypsin was set as enzyme, centrations of collagen (7569 ng/ml) while being continu- and up to two missed cleavages were allowed. Oxidation (M), ously stirred at 1000 rpm. The threshold concentration of acetylation (protein N-terminus), and HNE (1156.115 Da on collagen was defined as the concentration of collagen that in- lysine, K or histidine, H) were set as variable modification. duced a 50% increase in light transmission. The extent of Peptides were filtered with false discovery rate at 1%. The platelet aggregation was expressed in terms of percentage of mass spectrometry proteomics data have been deposited to change in light transmission 4 minutes after the addition of the ProteomeXchange Consortium via the PRIDE partner re- collagen. Blocking of CD36 or SRB1 receptor was achieved by pository with the data set identifier PXD013301 (DOI, preincubation with 10 ml of an anti-CD36 (Ab-CD36) or anti- 10.6019/PXD013301; username, [email protected]; SRB1 antibodies (dilution 1:500; Abcam) for 10 minutes at password, oMD4gyDT). 37°C before the incubation with HDL. Inhibition of SRC kinases was achieved by the preincubation with Naphthyl PP1 (final Molecular Modeling concentration 1 mM; Santa Cruz Biothechnologies, Dallas, Tostudy the accessibility of residues, the structure of truncated TX) for 10 minutes at 37°C before the incubation with human apo A-I (Protein Data Bank [PDB] code 1av1) was HDL. Aggregation values from lipoprotein assays were ex- downloaded from the PDB database.49 All observations were pressed as a percentage of the maximum aggregation induced performed using PyMOL software by representing the by the collagen alone (considered as 100%). Levels of throm- a-helices and histidine and lysine residues (colored in blue) boxane B2 (TxB2) was measured using an immunoassay ex- in CPK coloring to facilitate the study. The same method was pressed as a percentage of the maximum level induced by the achieved with the HDL model which was download from the collagen alone (considered as 100%; Cayman Chemical, Ann PDB database.50

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Statistical Analysis higher in the HDL of the CKD group (Figure 1A). MDA con- Data were expressed as median6interquartile range. All anal- centration was significantly higher in the HDL of the CKD yses were performed using GraphPad Prism version 6.0 group (Figure 1B), whereas the 8-isoprostane concentration (GraphPad software, La Jolla, CA). Simple comparisons was not significantly different (Figure 1C). Despite a nonsig- were made using the Mann–Whitney U test. Multiple compar- nificant difference in the concentration of tocopherols isons were made with Kruskal–Wallis test and, whenever ap- (Figure 1D), HDL lipids from CKD rabbits were more prone propriate, Dunn tests. Differences were considered as signifi- to oxidize than HDL from control animals; there was a signif- cant at P,0.05. icant difference from 70 minutes until the end of the experi- ment (P,0.05; Figure 1E).

RESULTS Platelet Aggregation Induced by HDL from Rabbits When human platelets were incubated with HDL from the General Characteristics of the CKD Rabbit Model CKD group, the median aggregation was 75% as compared Rabbits developed a significant renal insufficiency in the CKD with collagen alone; when incubated with HDL from the con- group; there was a significant difference (P,0.001) in the trol group, it was 30% (P,0.05 as compared with incubation median GFR between the control group and the CKD group with HDL from the CKD group; Figure 2A). Because HNE (258%; Table 1). CKD rabbits exhibited increased levels of adducts were increased in CKD HDL, we incubated control lipid peroxidation in the plasma as evidenced by the tenfold, rabbit HDL with 100 mM HNE; median platelet aggregation in significant increase in MDA concentration (P,0.001; Table 1). the presence of HNE-modified HDL was 85% (P,0.05 as Median AOA of the plasma was significantly (P,0.05) lower compared with incubation with HDL from the control group; in the CKD group compared with control (215%; Table 1). Figure 2A). Preincubation of platelets with Ab-CD36 restored Histologic examination found a diffuse fibrosis in the paren- the antiaggregant effect of the HDL from CKD rabbits and chyma in the CKD group as well as glomerulomegaly HNE-modifiedHDL(median,25%and22%,respectively; (Supplemental Figure 1). Figure 2, A and C). Preincubation of platelets with Ab-CD36 did not change significantly the aggregation induced by colla- HDL Oxidation Level and Oxidizability in CKD Rabbits gen (median, 95% versus 100%; NS). HDLfromCKDrabbitshadsignificantly higher levels of To investigate the effect of total lipoproteins (HDL, LDL, HNE-Michael adducts on proteins (P,0.05; Figure 1A). In and triglyceride-rich lipoproteins; LPP) of CKD animals, contrast, HHE adducts on proteins were not significantly platelets were incubated with a lipoprotein mix resulting

A C * Control 6000 15 CKD ns E 4000 10 0.3 AU 2000 ns 5 8-isoprostane

(pg/mg of proteins) 0.2 0 0 p < 0,05 HNE-adducts HHE-adducts Control CKD

0.1

B D CKD 4 800 ns * Control Conjugated dienes formation, AU 0.0 3 600 0 70 100 200 300 Time, min 2 400

1 Tocopherols, 200 MDA (nmol/mg) pmol/mg proteins 0 0 Control CKD Control CKD

Figure 1. Oxidation of HDL particles from CKD rabbits. HNE adducts were increased in HDL from (A) CKD rabbits as well as (B) MDA concentration. No difference was observed with (C) 8-isoprostane and (D) tocopherol concentrations. (E) HDL from CKD rabbits were more prone to oxidation after a copper-induced oxidation as described. n58 and 9, CKD and control, respectively; *P,0.05, Mann–Whitney test. Data are expressed as median and interquartile range.

JASN 31: ccc–ccc, 2020 Carbonylation of HDL in CKD 5 BASIC RESEARCH www.jasn.org from a single ultracentrifugation. The median aggregation in total cholesterol, LDL cholesterol, and HDL cholesterol than the CKD LPP group was 98% versus 21% in the control group the controls (P,0.05). There was a higher level of HNE ad- (P,0.05; Figure 2B). Platelet aggregation in the presence of ducts in HDL from patients on HD than in controls HNE-modified LPP was 99% (P,0.05 compared with control (Figure 3A). To map sites on proteins susceptible to carbon- LPP group). Preincubation of platelets with Ab-CD36 restored ylation, HDL from control participants and patients on HD the antiaggregant effect of the LPP from CKD rabbits and were digested with trypsin; digested peptides were separated HNE-modified LPP (median, 23% and 11%, respectively; by liquid chromatography, and peptides were analyzed for Figure 2B). HNE-adduct levels in HNE HDL and LPP were carbonylation with tandem mass spectrometry. We then ex- higher than in control using an immunoblot (Figure 2D). pressed the ratio of amount of HNE in the HD patient sample versus the mean amount of control patients. A higher ratio Carbonylation of HDL from Patients on HD (greater than one) of HNE-Michael adduct was detected on 48 Nine controls were compared with nine nondiabetic patients amino acids (lysine or histidine) from eight constitutive pro- on HD, the main characteristics of whom are presented in teins of HDL from patients on HD (Tables 2 and 3). The Supplemental Table 1. Patients on HD had significantly lower majority were located on apo A1 and concerned various

* * * * * A * * B 100 * 100

50 50 % of aggregation % of % of aggregation % of

0 0

Collagen ++++++++ Collagen ++++++++ CD36 Ab - + - + - + - + CD36 Ab - + - + - + - + Control HDL --++---- Control LPP --++---- CKD HDL ----++-- CKD LPP ----++-- HNE HDL ------++ HNE LPP ------++

CDCollagen 1.5×107 T4 min

Collagen + CKD HDL 1.4×107

T4 min 1.3×107 Aggregation Collagen + Control HDL Baseline T4 min 7

HNE Michael adducts (UA) 1.2×10 Collagen T0 Time, min Control CKD HNE Dot Blot

Figure 2. CKD is responsible for impaired antiaggregant properties of HDL in rabbits. (A) CKD HDL exhibited blunted antiaggregative properties compared with control HDL. Control HDL modiﬁed by an incubation overnight with HNE (HNE HDL) solution exhibited similar blunted properties to CKD HDL compared with control HDL. Preincubation with Ab-CD36 restored the CKD and HNE HDL antiaggregant properties like control HDL. (B) Typical aggregation curves obtained with collagen, control HDL, and CKD HDL. Col- lagen aggregation level is considered as 100%. The same results were observed with a lipoprotein mix containing triglyceride-rich lipoproteins (VLDL and chylomicrons), LDL, and HDL (LPP) from control and CKD rabbits. CKD LPP exhibited blunted antiaggregative properties compared with control LPP. (C) Control LPP modiﬁed by an incubation overnight with HNE solution (HNE LPP) exhibited similar properties to CKD LPP. Preincubation with Ab-CD36 restored the CKD and HNE LPP antiaggregant properties like control LPP. (D) HNE HDL had increased the level of HNE adducts compared with control HDL. n58 and 9, CKD and control, respectively. *P,0.05, Mann–Whitney test. Data are expressed as median and interquartile range.

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AB 10 20 30 40 50 7 3.3×10 MKAAVLTLAV LFLTGSQARH FWQQDEPPQS PWDRVKDLAT VYVDVLKDSG

* 60 70 80 90 100

RDYVSQFEGS ALGKQLNLKL LDNWDSVTST FSKLREQLGP VTQEFWDNLE 3.2×107 110 120 130 140 150 KETEGLRQEM SKDLEEVKAK VQPYLDDFQK KWQEEMELYR QKVEPLRAEL 7 160 170 180 190 200

AU 3.1×10 QEGARQKLHE LQEKLSPLGE EMRDRARAHV DALRTHLAPY SDELRQRLAA

210 220 230 240 250 3.0×107 RLEALKENGG ARLAEYHAKA TEHLSTLSEK AKPALEDLRQ GLLPVLESFK

260

VSFLSALEEY TKKLNTQ 2.9×107 Control HDL HD HDL Important to stabilize lipid-free apo-A1 structure in solution Important for cholesterol efflux activity Dot Blot Important for aggregant properties Important for lipid affinity

C Sequence Modifications Theo. MH+ [Da] Abundances (Control) Abundances (HD) Abundances Ratio P-Value [R].QGLLPVLESFKVSFLSALEEYTK.[K] 1xHNE [K11] 2754.52156 28.2 171.8 5.3 0.009

Peptide [241-262] : MH+ : 2598.4056 Th - MSMS on m/z 1299.7064 (2+ ; -0.37 ppm) T: FTMS + cNSI d Full ms2 [email protected] [100.0000-2670.0000] 299.1720 100 + b 95 3 90 b b b b b b b b b b b 85 2 3 4 5 6 7 8 9 10 11 12 80 Q-G-L-L-P-V-L-E-S-F-K-V-S-F-L-S-A-L-E-E-Y-T-K y y y y y y y y y y y y y y y y y y y 75 20 19 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 70 65 60 55

50 1291.6967 45 40 Relative Abundance 35 1094.5874

2+ 30 + y19 b4 25 169.1339 y + y + y + + 3 4 11 + y2 b2 540.2670 20 411.2218 248.1607 591.3525 y + y + y + 5 + 8 12 y + y + y7 15 19 15 669.3100 + 940.4656 1386.7190 + y + y13 2188.1511 6 853.4334 y 2+ + 1748.9287 + 9 y y y16 10 + b + 782.3946 20 10 1514.8134 y + y + y1 6 1053.5471 14 1878.9523 17 + + 1151.1279 b5 + b8 + 1662.8798 1992.0461 b7 + b12 5 b11 1569.8215 2042.0938 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 m/z

Peptide [241-262] : HNE Modification : MH+ : 2754.5001 Th - MSMS on m/z 689.3805 (4+ ; -7.78 ppm) T: FTMS + c NSI d Full ms2 [email protected] [100.0000-2835.0000] 100 169.1335 b1 b2 b3 b4 b6 b8 b10 Q-G-L-L-P-V-L-E-S-F-KHNE-V-S-F-L-S-A-L-E-E-Y-T-K y y y y y y y y y y 90 19 18 17 16 15 14 13 7 3 1

b + 1 797.4038 80 100 797.4038 95 197.1284 90 85 + 70 b3 80 75 910.4874 299.1713 70 782.0880 65 1068.5575 60 60 55 910.4874 2+ y17 50 3+ y19 1075.0724 394.2450 45 50 1068.5575 981.5261 40 782.0880 892.4780 35 30 832.4950 2+ 25 y16 Relative Abundance 40 668.3611 y 2+ 2+ 3+ 3+ 13 y15 1018.0241 y 20 y17 19 2+ 776.0845 2+ 953.5032 y17 y 15 716.3857 3+ 14 y 837.4472 18 b + 10 749.3995 819.4418 966.4910 1051.5525 10 + 981.52561 + 900.5195 938.5090 b2 590.3659 y7 724.3631 1084.61 30 5 853.4180 870.4711 920.5009 1009.5211 539.3179 0 750 800 850 900 950 1000 1050 1100 y + 1 m/z 282.1427 367.2335 832.4950 20 688.7109 455.7478 2+ y18 y 2+ b + 16 y 2+ 4 3+ y 2+ 1124.0952 19 y17 15 y 2+ 1172.6222 10 y 3+ 13 18 y142+ 1443.7228 + 1543.7804 b6 1275.6992 + + y3 b10 1237.6403 0 100200 300 400 500 600 700 800 900 10001100 1200 1300 1400 1500 1600 1700 m/z

Figure 3. HNE adducts are increased in HDL from patients on HD compared with controls. (A) Immunoblotting (dot blot) of HNE- Michael adducts were performed as described in the Methods section. Identiﬁcation of increased HNE-Michael adduct on lysine and histidine residues of HDL from patients on HD by liquid chromatography–tandem mass spectrometry assay (MSMS). HDL was reduced, alkylated, and digested with trypsin. (B) Sequence of apo A1 (accession number, P02647-1) indicating the position of the adducts (bold characters) and the sequences of interest (highlighted and framed). Typical MSMS spectra from unmodiﬁed peptide (upper spectrum)

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Table 2. General characteristics of controls and patients on HD and PD et al. (Figure 4B)50 led to the following Characteristics Control (N515) HD (N525) PD (N520) observations: some histidine and lysine General characteristics residues are not involved in interactions Age, yr 46 (36–58) 56 (43–73) 67 (56–84)a,b with lipids and are located at the external Gender, % male 56 43 56 part of HDL. These residues that can re- BMI, kg/m2 26 (24–28) 24 (20–26) 25 (23–28) act with HNE may not affect the binding Comorbidities of apo A-I with lipids but with other pro- HT, n (%) 3 (21) 17 (74) 19 (95) teins involved in HDL metabolism (see Stroke, n (%) 0 (0) 2 (9) 1 (5) Figure 3B, residues K47, K83, H216, CHD, n (%) 0 (0) 5 (22) 3 (15) K250 and K262); in contrast, some his- Cardiopathy, n (%) 0 9 (39) 8 (40) tidine and lysine residues (residues K64, PVD, n (%) 0 4 (17) 3 (15) K130, H179, H186, and H223) interact Therapies, n tightly with lipids in the internal side of Statins 0 6 5 PI 0 5 6 the complex. In this case, it can be pro- fi RASi 1 3 11 posed that these modi cations may alter b-Blockers 1 5 4 the ability of apo A-I to bind lipids. Over- CCB 1 1 6 all, this molecular modeling study shows Biologic parameters that the reaction of lysine and histidine Urea, mmol/L 5.4 (4.4–7.5) 17.1 (13.3–19.8)a 18.8 (16.1–22.6)a residues leading to structural modifica- Creatinine, mmol/L 73 (69–85) 702 (506–862)a 575 (422–758)a tionsmayoccurrandomlybutmaybe 2 mGFR, ml/min per 1.73 m 94 (86–96) ——critical for lipid and protein binding, – – a – b Total cholesterol, mg/dl 208 (189 237) 149 (113 180) 209 (155 232) depending on the residue which is – – a – b LDL cholesterol, mg/dl 139 (105 158) 71 (54 103) 125 (81 159) modified. HDL cholesterol, mg/dl 59 (53–66) 41 (30–47)a 45 (36–62)a Triacylglycerols, mg/dl 91 (64–116) 119 (88–173)a 149 (118–207)a CRP, mg/L 1.9 (0.5–3.5) 3.3 (1.9–8.0) 3.7 (1.7–5.8) Platelet Activation and Data are expressed as median (interquartile range). eGFR measured by the CKD Epidemiology Col- Aggregation Phenotype of HDL laboration formula. BMI, body mass index; HT, hypertension; CHD, coronary heart disease; PVD, from Patients on HD and PD and peripheral vascular disease; PI, platelet inhibitor; RASi, renin-angiotensin system inhibitor; CCB, Effects of Carbonylation calcium-channel blocker; mGFR, measured GFR by iohexol clearance; CRP, C-reactive protein. aP,0.05 versus controls. Because the HD procedure per se can lead bP,0.05 versus HD, Mann–Whitney U test. to enhanced oxidative and carbonylated stress, we included patients on PD to avoid any effects resulting from bio- important functional sites of the protein (Figure 3B, incompatibility of HD devices. A total of 15 healthy controls Supplemental Figure 2). Lys 250, located in the C terminal were compared with 25 nondiabetic patients on HD and 20 part of apo A1, was the most frequently modified site in pa- patients on PD, the main characteristics of whom are presen- tients on HD (5.3-fold increase compared with controls, ted in Table 2. Patients on HD had significantly lower total P,0.01; Figure 3C). cholesterol, LDL cholesterol, and HDL cholesterol than the controls (P,0.05). Platelet activation was measured with the In Silico Modeling of HNE Adduction onto apo A1 production of TxB2 and expressed as a percentage of the To gain insights into the accessibility of histidine and lysine amount found in platelets activated with collagen. When hu- residues of apo A1, a molecular modeling study was achieved man platelets were incubated with HDL from the HD group, with the lipid-free structure of the truncated protein the median activation was 147% as compared with collagen (Figure 4A).49 Histidine and lysine residues mentioned in alone; when incubated with HDL from the control group it the primary sequence of amino acids—namely K83, K130, was 75% (P,0.05 as compared incubation with HDL from the H179, H186, H216, H223, K226, K250, and K262—were ex- HD group; Figure 5A). When human platelets were incubated amined and were found to be readily accessible and susceptible with HDL from the PD group, the median activation was to react with HNE (not available for the H20, K47, and K64 221% as compared with collagen alone (P,0.05 as compared residues in the truncated protein). Therefore, the modification with incubation with HDL from the control group, P5NS as of these residues may occur in a random event on free apo A1. compared with incubation with HDL from the HD group; Careful examination of the model of HDL proposed by Wu Figure 5A). Because HNE adducts were increased in HD

and modiﬁed peptide (lower spectrum). (C) The abundance of HNE adduct of Lys 250 in HD HDL was 5.3-fold higher than in controls (P,0.01). n59 and 9, HD and control HDL, respectively. * P,0.05, Mann–Whitney test. Data are expressed as median and interquartile range.

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Table 3. List of HNE-adducted amino acids in HDL constitutive proteins from patients on HD Protein Name Protein Label %a Site of Adduction Abundance Ratio (HD 1–9, respectively)b apo B100 APOB 0.04 K3689 0.293 0.825 0.468 4.099 8.148 1.488 1.626 3.606 0.322 K4498 ————4.421 ——2.339 — Serum albumin ALB 1.15 H152 2.646 5.05 0.373 — 0.036 0.072 1.263 0.874 6.837 K160 or H152 2.956 6.049 0.229 — 0.295 — 0.718 0.405 12.994 K347 — 1.452 — 0.879 — 0.582 1.767 2.442 1.966 H170 1.463 ——0.919 — 0.178 0.957 —— K581 0.861 1.148 —— — —0.668 1.944 2.204 K198 1.715 ————2.864 ——1.28 apo A1 APOA1 4.50 H20 0.516 0.863 1.131 2.298 1.414 1.284 1.133 1.109 0.556 H179 2.448 2.756 5.133 1.117 0.995 1.306 0.316 0.802 0.853 H186 2.646 2.508 4.401 0.613 0.806 0.93 0.168 1.221 1.767 H223 0.456 0.549 1.471 0.101 0.125 0.188 — 0.136 — H217 1.342 0.939 3.216 0.039 0.175 0.266 — 0.14 — K36 0.89 1.362 1.181 1.64 0.996 1.36 1.873 1.863 1.128 K250 1.423 3.189 0.548 4.329 31.087 1.035 3.154 30.549 2.522 K130 0.332 1.236 0.856 ———1.018 — 0.728 K47 0.596 1.345 1.237 2.083 0.861 1.211 2.596 2.217 1.557 K83 0.393 — 0.943 1.004 0.559 0.79 2.181 2.642 2.131 K64 0.571 1.139 1.079 1.643 1.119 1.543 1.403 1.616 0.935 K262 — 10.052 — 0.757 7.534 — 0.442 42.042 — apo(a) LPA 0.02 K215 — 100 —— — ——100 — apo A2 APOA2 1 K26 — 1.983 — 0.134 ——0.963 0.29 0.689 Keratin, type I, cytoskeletal 10 KRT10 0.17 K362 2.598 3.076 3.419 1.44 0.596 1.462 0.778 2.66 1.595 Trypsin-3 PRSS3 0.32 H153 1.292 0.7 1.056 1.332 0.827 5.646 0.972 0.914 0.906 UDP-glucose:glycoprotein UGGT2 0.06 H1388 1.46 1.385 3.62 3.525 4.433 2.838 5.1 11.999 1.877 glucosyltransferase 2 K, lysine; —, not found; H, histidine; UDP, uridine diphosphate. aPercentage of potential sites of adduction regarding all of the amino acids of the protein sequence. bAbundance ratio was calculated against the mean of the nine controls.

HDL, we incubated five healthy donors’ HDL with 100 mM only the 100 mM solution of HNE led to a significant alteration HNE; median platelet activation in the presence of HNE- of the antiaggregant properties of HDL (median, 5% at modified HDL was 123% as compared with collagen alone, 100 mM versus 98%, 99%, and 99% at 1, 10, and 50 mM, re- when incubated with HDL from the control group it was 29% spectively; P,0.05; Figure 5D). Immunoblotting found a (P,0.05 as compared with incubation with HDL from the dose-dependent increase of HNE adducts in HDL incubated healthy donor group; Figure 5B). with HNE (1–100 mM; Supplemental Figure 4).The median As we performed aggregation assays in rabbits, we chose levels of HNE adducts in HNE-modified HDL were similar to nine control and nine patients on HD (characteristics in those of patients on HD and PD (see Supplemental Figure 5). Supplemental Table 1) and performed platelet aggregation assays. When human platelets were incubated with HDL from Activation Is Mediated by the Phosphorylation of c-Jun the HD group, the median aggregation was 50% as compared N-Terminal Kinase through a CD36 and SRC Kinase with collagen alone; when incubated with HDL from the con- Pathway trol group it was 19% (P,0.05 as compared with incubation To get insight into the potential pathways involved in the ef- with HDL from the HD group; Figure 5C). We found a good fects described herein, the effect of CD36, SRB1 blockade, and correlation between percentage of activation with TxB2 assay SRC kinase pharmacologic inhibitor were tested. Preincuba- and percentage of aggregation (r250.39, P,0.05; tion of platelets with Ab-CD36 significantly decreased their Supplemental Figure 3). Because HNE adducts were increased activation as measured by TxB2 levels when incubated with in HD HDL, we incubated healthy donors HDL with 100 mM HDL from control, HD, PD, and HNE-modified HDL (me- HNE; median platelet aggregation in the presence of HNE- dian, 39%, 41%, 22%, and 49%, respectively; P,0.05, as com- modified HDL was 170% (P,0.05 as compared with incuba- pared with incubation without the antibody; Figure 6A). tion with HDL from the control group; Figure 5C). P-selectin expression was also lowered by the preincubation Because there was an interindividual variability of platelet of platelets with Ab-CD36 for HDL from control, HD, PD, response to HD HDL, we tested several concentrations of HNE and HNE-modified HDL (median, 243%, 230%, 242%, for the modification of control HDL from healthy donors and 227%, respectively; P,0.05 as compared with incubation (1, 10, 50, and 100 mM). A threshold effect was found because without the antibody; Figure 6B). Preincubation of platelets

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A 22%, 46%, 24%, and 22%, respectively; P,0.05 as compared with incubation without the inhibitor; Figure 6A). P-selectin expression was also decreased by the preincubation of platelets with Naphthyl PP1 for HDL from control, HD, PD, and HNE- modiﬁed HDL (median, 248%, 248%, 253%, and 214%, respectively; P,0.05 as compared with incubation without the inhibitor; Figure 6B). We conﬁrmed that intraplatelet phosphorylation of c-Jun N-terminal kinase (JNK) was reduced by blocking of CD36 and SRC kinases, whereas blocking SRB1 increased its phosphorylation(datanotshown). B

DISCUSSION

This study confirms that HDL isolated from patients with CKD exhibit impaired biologic functions that may participate in the onset of cardiovascular disease in this population. The work emphasizes the effect of carbonylation from long-chain n-6 fatty acids (HNE adducts) on the antiaggregative properties of HDL. This adduction onto the protein component of Figure 4. Structure of truncated human apo A-I (pdb code 1av1) the HDL particle was responsible for a deep and significant revealed several accessible sites for post-translational modifica- alteration of the antiaggregant properties of HDL in CKD. tions by 4-HNE. Four molecules are associated with their hydro- Combined with all of the other impaired functions of HDL, phobic faces to form an antiparallel four-helix bundle. (A) Lysine this could be a potent contributor to the increased cardiovascu- and histidine residues possibly modified by HNE are colored in lar morbidity and mortality in CKD3 and the failure of statins in cyan with CPK representation. (B) Model of HDL:phospholipids patients in HD.27,28 Moreover, a significant part of this patho- (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) are colored logic phenotype was mediated by a CD36-dependent pathway in yellow and cholesterol in magenta. Lysine and histidine resi- because its blockade restored normal antiaggregant properties. dues possibly modified by HNE are colored in cyan with CPK coloring representation (pdb code 3k2s). Furthermore, CKD per se was responsible for a large part of this carbonylation because these adducts were obtained in a rabbit CKD model without other cardiovascular risk factors. with Ab-CD36 restored the antiaggregant effect of the HDL The rabbit model is of interest for cardiovascular research from HNE-modified HDL (median, 12%; P,0.05 as com- (e.g., atherosclerosis, lipid metabolism) because these animals pared with incubation without the antibody; Supplemental have a lipoprotein metabolism much closer to humans than Figure 6). Preincubation of platelets with Ab-CD36 did not mice or rats due to the expression of the cholesteryl ester significantly change the aggregation induced by collagen (me- transfer protein gene. The CKD rabbits, despite having a lower dian, 107% versus 100%; NS; Supplemental Figure 6). body weight, developed a metabolic disturbance because their Preincubation of platelets with an anti-SRB1 antibody sig- plasma cholesterol and glucose levels were significantly higher. nificantly increased their activation as measured by TxB2 lev- The median measured GFR reduction in CKD animals corre- els when incubated with HDL from control, HD, PD, and sponded approximately to stage 3 CKD in humans, and results HNE-modified HDL (median, 385%, 178%, 288%, and of oxidative stress assays (MDA and AOA) were consistent 758%, respectively; P,0.05 as compared with incubation with values observed in a human CKD population,5 which without the antibody; Figure 6A). P-selectin expression was supports the appropriateness of the model. However, a natural also increased by the preincubation of platelets with an anti- history of 3 weeks is unlikely to lead to the same modifications SRB1 antibody for HDL from control, HD, PD, and HNE- as several years of interaction between cardiovascular risk fac- modified HDL (median: 135%, 193%, 154%, and 188%, tors and uremia in humans. Nevertheless, we found an in- respectively; P,0.05 as compared with incubation without the crease in HNE adducts and the impaired antiaggregant prop- antibody; Figure 6B). Preincubation of platelets with anti- erties of HDL in patients on HD and PD, rendering the animal SRB1 did not significantly alter the aggregation induced by findings relevant. collagen (data not shown). The results of this study suggest that CKD per se may trigger Preincubation of platelets with Naphthyl PP1, a potent a strong oxidation of HDL particles. Interestingly, HNE ad- pharmacologic inhibitor of SRC kinases, decreased their acti- ducts were both increased in the HDL of CKD rabbits and vation as measured by TxB2 levels when incubated with HDL patients on HD and PD. This can be due to a loss of antioxi- from control, HD, PD, and HNE-modified HDL (median, dant capacity of HDL particles in CKD because HDL from

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A 650 * B 600 250 * 200

* 150

400 100

% of activation (T×B2) 0 200 Collagen Collagen % of activation (T×B2) + + 100 Control HDL HNE HDL

0 D 100 Collagen Collagen Collagen + + + Control HD PD HDL HDL HDL 50 C 250 * 200 % of anti-aggregation * 150 0 Collagen +++++ 100 Control HDL + ---- * HNE 1µM HDL - + --- % of aggregation 50 HNE 10µM HDL -- + -- HNE 50µM HDL --- + - 0 HNE 100µM HDL ---- + Collagen Collagen Collagen Collagen + + + Control HD HNE Dot Blot HDL HDL HDL

Figure 5. HDL from patients on PD and HD exhibited impaired antiaggregant properties. (A) Regarding levels of TxB2, HD and PD HDL triggered overactivation of platelets compared with control HDL (n515 for control, n525 for HD group, and n520 for PD group). (B) The same observations were found for healthy volunteers’ HDL modified by an incubation overnight with HNE (HNE HDL, n55for each group). (C) HD, PD, and HNE HDL exhibited blunted antiaggregant properties compared with control HDL (n59 for control and HD groups, n55 for HNE group). (D) We found a threshold effect of HNE modification on HDL because only a 100 mMsolutionofHNE permitted the significant alteration of the antiaggregant properties of control HDL (n54 for each group). *P,0.05, Mann–Whitney test and Kruskal–Wallis test (in D). Data are expressed as median and interquartile range.

CKD rabbits were more sensitive to copper-induced oxida- on HD and CKD rabbits, suggesting a blunted antithrombotic tion. This is in accordance with data from Holzer et al.24 profile of uremic HDL. To exclude that the blunted antiaggre- who reported in patients on PD and HD a profound modifi- gant properties in patients on HD did not only result from the cation of HDL structure, in particular a lower concentration of HD procedure per se,weconfirmed this profile in patients on paraoxonase. Moreover, the short natural history of CKD in PD. This strengthened the role of CKD and especially ESKD in the model used here suggests that these modifications occur the pathologic properties of HDL. This is consistent with the during the early stages of the disease. These results are consis- increase in the incidence of thrombotic events in CKD, espe- tent with that reported by Shroff et al.25 and Kaseda et al.51 in cially in patients on HD27,28,52,53 and with a higher platelet pediatric patients with CKD, who can be considered as close to reactivity in CKD.54–57 However, we cannot totally rule out the model used because of a short natural history of CKD and a the role of age as a confounding factor in the different aggre- lower number of CKD-associated comorbidities. gation behavior of HDL because patients on PD were older The results regarding platelet activation and aggregation than the patients on HD and controls. The pathophysiologic are consistent with the altered properties of HDL in CKD; pathways of the platelet aggregation profile in CKD has only we observed a significant increase in the activation of platelets been studied for LDL; Holy et al.31 found a proaggregant profile incubated with HDL from patients on HD and PD and a of LDL in CKD that was mostly explained by carbamoylation of twofold reduction in antiaggregation with HDL from patients these proteins. Moreover, this study highlighted the role of the

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A 10000 Without blocker Ab anti-CD36 Ab anti-SRB1 Naphtyl PP1

1000

* †

100 $ $ * † †

% of activation (TxB2) *

1 Control HDL HD HDL PD HDL HNE HDL

B 150 Ab anti-CD36 Ab anti-SRB1 * Naphtyl PP1

100

0 Variation of activation (%, P-Selectin)

-50

* *

-100

HD PD HD PD HD PD CTL HNE CTL HNE CTL HNE

Figure 6. The impaired antiaggregant properties of HDL in patients on PD and HD and healthy volunteers’ HDL modiﬁed by an incubation overnight with HNE (HNE HDL) is mediated by CD36 binding and SRC kinase–mediated pathways. (A) Regarding levels of TxB2, the preincubation of platelets with an antibody against CD36 receptor (Ab anti-CD36) signiﬁcantly lowered the activation of platelets exposed with control (CTL), HD, PD, and HNE compared with incubation without the antibody. The preincubation of platelets

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H 4-hydroxy-2-nonenal adduct P Phosphorylation H HH + Activation H CD36 Platelet - Inhibition H SRC kinases CKD HDL P P + JNK + Activation

H HH - H H SR-B1 Inhibition CKD HDL

Native HDL

Figure 7. HDL from patients with CKD and rabbit (CKD HDL) exhibited blunted antiaggregant properties by interaction with CD36 receptor. Carbonylation by HNE of proteins from HDL was found to be part of this pathologic behavior. Activation of the platelets in response to this binding was through a SRC kinase–mediated pathway. SRC kinase activation resulted with an increase of phosphorylated JNK (pJNK). Native and CKD HDL apo A1 binds SRB1 and inhibits platelet activation. lectin-like oxidized LDL receptor 1 in the onset of this profile; a involved in the aggregant properties of HDL. Indeed, native receptor mainly involved in LDL-mediated pathophysiologic and mildly oxidized HDL binds ApoER2 and SRB1 scavenger pathways. The proagreggant profile of oxidized LDL has been receptor, inducing an intracellular downregulation of platelet described by Chen et al.32 to be mediated by CD36 and activation pathways. Conversely, heavily oxidized HDL binds this receptor is also known to bind heavily modified CD36 and platelet activation is upregulated.58,61 Thus, we can HDL58,59 and oxidized albumin.60 The results of this study hypothesize that the adduction induced by the incubation suggest a strong implication in the blunted antiaggregant pro- with HNE at a concentration ,100 mM caused light or mild file of the CD36-mediated pathway because blocking modifications of the protein component of HDL insufficient CD36 restored the antiaggregant properties of CKD and to bind CD36. HNE adducts to the protein component of HNE-modified HDLs. Furthermore, this study suggests the HDL particles were associated with proaggregative properties involvement of SRC kinase–mediated pathways in the platelet of HDL. Interestingly, the mass spectrometry assay specifically activation process in response to CKD and HNE-modified identified such HNE adducts on lysines and histidines from HDLs because the use of a specific pharmacologic inhibitor eight constitutive proteins of HDL and especially on apo A1 of (Naphthyl PP1) also restored the antiaggregant properties of patients on HD. This apo is widely responsible for the anti- these HDLs. Finally, we confirmed that the binding of HDL aggregant properties of HDL33 and several modifications were onto the CD36 receptor resulted in a significant increase in JNK locatedonkeypartsoftheproteininvolvedincholesterol phosphorylation as demonstrated by Chen et al.32 for oxidized efflux, platelet aggregation, and lipid binding. LDL. A schematic summary of these pathways is summed up in Post-translational modifications of proteins represent a Figure 7. wide spectrum of potentially toxic modifications such as car- The study also suggests that there is a threshold effect of bamoylation, generations of advanced glycation end products, HNE adduction on aggregative properties of HDL because the chlorination, or nitration. For example, carbamoylation of incubation of control HDL with HNE in concentrations LDL particles was responsible for proaggregant properties of ,100 mM did not modify the antiaggregant profile of HDL. platelets in patients with CKD.31 Despite a strong effect of the This could be explained by the multiple receptors that are carbonylation by HNE described herein, we cannot exclude with an antibody against SRB1 receptor (Ab anti-SRB1) significantly increased the activation of platelets exposed with CTL, PD, and HNE compared with incubation without the antibody. The preincubation of platelets with a pharmacologic inhibitor of SRC kinases (Naphthyl PP1) significantly lowered the activation of platelets exposed to CTL, HD, PD, and HNE compared to incubation without the inhibitor. (B) In a FACS assay, activation of platelets were determined by the expression of P-selectin. The preincubation of platelets with Ab-CD36 and Naphthyl PP1 significantly decreased the activation of platelets exposed to CTL, HD, PD, and HNE HDL compared with the samples without the antibody/inhibitor. The preincubation of platelets with Ab anti-SRB1 significantly increased the activation of platelets exposed to CTL, HD, PD, and HNE HDL compared with the samples without the antibody/inhibitor. n55 for each group. † *P,0.05 compared with control HDL without blocker; $P,0.05 compared with HD HDL without blocker; P,0.05 compared with PD HDL without blocker; *P,0.05 compared with HNE HDL without blocker; Mann–Whitney test. Without blocker, without either Ab- CD36, Ab anti-SRB1, or Naphthyl PP1.

JASN 31: ccc–ccc, 2020 Carbonylation of HDL in CKD 13 BASIC RESEARCH www.jasn.org that other post-translational modifications could also contrib- Supplemental Methods. ute to the proaggregant properties of HDL. However, further Supplemental Table 1. General characteristics of hemodialysis and studies should be dedicated to explore the specific role of other control patients for aggregation assay. kinds of post-translational modification of lipopoproteins. To Supplemental Figure 1. Histological examinations of rabbit conclude, we describe herein for the first time the impaired kidneys. antiaggregant properties of HDL in CKD. To improve cardio- Supplemental Figure 2. Examples of modified amino-acid MS/MS vascular outcomes in CKD, future studies need to focus on spectra. HDL quality as well as quantity. Supplemental Figure 3. Spearman correlation of TxB2 assay and aggregation assay. Supplemental Figure 4. Spearman correlation of the amount of 4- ACKNOWLEDGMENTS HNE adducts on in-vitro modified HDL. Supplemental Figure 5. Immunoblotting of 4-HNE adducts me- The authors gratefully acknowledge Philip Robinson (Department of dian levels of HNE-modified HDL, HD, PD and control HDL. Clinical Research and Innovation, University Hospital of Lyon) for Supplemental Figure 6. Aggregation levels of HNE-modified HDL help in manuscript preparation. The authors acknowledge D. Yi and versus control and CD36 blockade effect. E. Hoibian (CarMeN Laboratory) for their help during the experiments. The authors acknowledge Dr. M. Rabeyrin (Department of Pathology, E. Herriot Hospital, Lyon, France) for preparation of the REFERENCES histological samples and Dr A. Varennes (Department of Bio- chemistry, E. Herriot Hospital, Lyon, France) for urea and creatinine 1. Go AS, Chertow GM, Fan D, McCulloch CE, Hsu C-Y: Chronic kidney measurements. disease and the risks of death, cardiovascular events, and hospitaliza- tion. NEnglJMed351: 1296–1305, 2004 Dr. Florens, Dr. Juillard, Dr. Moulin, and Dr. Soulage conceptu- 2. Schiffrin EL, Lipman ML, Mann JFE: Chronic kidney disease: Effects on alized and designed the study; Dr. Barba, Dr. Boulet, Dr. Calzada, Dr. the cardiovascular system. Circulation 116: 85–97, 2007 Florens, Dr. Guillot, Dr. Lemoine, Ms. Roux, and Dr. Soulage per- 3. Tonelli M, Muntner P, Lloyd A, Manns BJ, Klarenbach S, Pannu N, et al.: formed the experiments; Dr. Delolme and Dr. Page performed Alberta Kidney Disease Network: Risk of coronary events in people with the mass spectrometry analysis; Dr. Florens and Dr. Soulage analyzed chronic kidney disease compared with those with diabetes: A population-level cohort study. Lancet 380: 807–814, 2012 the data and wrote the manuscript; Dr. Guebre-Egziabher edited the 4. Maduell F, Moreso F, Pons M, Ramos R, Mora-Macià J, Carreras J, et al.; manuscript; and Dr. Boulet, Dr. Calzada, Dr. Florens, Dr. Guebre- ESHOL Study Group: High-efficiency postdilution online hemodiafil- Egziabher, Dr. Juillard, Dr. Laville, Dr. Lemoine, Dr. Moulin, Dr. tration reduces all-cause mortality in hemodialysis patients [published Poux, and Dr. Soulage revised the manuscript. Dr. Florens is the correction appears in JAmSocNephrol25: 1130, 2014]. JAmSoc – guarantor of this work and, as such, had full access to all of the data in Nephrol 24: 487 497, 2013 5. Kuchta A, Pacanis A, Kortas-Stempak B, Cwiklinska A, Zie˛tkiewicz M, the study and takes responsibility for the integrity of the data and the Renke M, et al.: Estimation of oxidative stress markers in chronic kidney accuracy of the data analysis. disease. Kidney Blood Press Res 34: 12–19, 2011 6. Dounousi E, Papavasiliou E, Makedou A, Ioannou K, Katopodis KP, Tselepis A, et al.: Oxidative stress is progressively enhanced with ad- – DISCLOSURES vancing stages of CKD. Am J Kidney Dis 48: 752 760, 2006 7. Aveles PR, Criminácio CR, Gonçalves S, Bignelli AT, Claro LM, Siqueira SS, et al.: Association between biomarkers of carbonyl stress with in- All authors have nothing to disclose. creased systemic inflammatory response in different stages of chronic kidney disease and after renal transplantation. Nephron Clin Pract 116: c294–c299, 2010 FUNDING 8. Vanholder R, Massy Z, Argiles A, Spasovski G, Verbeke F, Lameire N; European Uremic Toxin Work Group: Chronic kidney disease as cause of cardiovascular morbidity and mortality. Nephrol Dial Transplant 20: Dr. Florens was supported by a Agence Régionale de Santé de Rhône Alpes (ARS) 1048–1056, 2005 “ ” grant Année Recherche, Hospices Civils de Lyon (University Hospital of Lyon), 9. Dou L, Sallée M, Cerini C, Poitevin S, Gondouin B, Jourde-Chiche N, and by Société Française Néphrologie Dialyse et Transplantation (French Society of et al.: The cardiovascular effect of the uremic solute indole-3 acetic “ – ” fi Nephrology) grant IRCT Dialyse. The authors acknowledge the nancial sup- acid. J Am Soc Nephrol 26: 876–887, 2015 port from ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et 10. Ito S, Osaka M, Edamatsu T, Itoh Y, Yoshida M: Crucial role of the aryl de la Santé; National Alliance for Life Sciences and Health) within the framework of hydrocarbon receptor (AhR) in indoxyl sulfate-induced vascular in- the cancer plan for the founding of the Orbitrap mass spectrometer. flammation. J Atheroscler Thromb 23: 960–975, 2016 11. Lin C-J, Liu H-L, Pan C-F, Chuang C-K, Jayakumar T, Wang T-J, et al.: Indoxyl sulfate predicts cardiovascular disease and renal function de- SUPPLEMENTAL MATERIAL terioration in advanced chronic kidney disease. Arch Med Res 43: 451–456, 2012 12. Velasquez MT, Ramezani A, Manal A, Raj DS: Trimethylamine N-oxide: This article contains the following supplemental material online at The good, the bad and the unknown. Toxins (Basel) 8: E326, 2016 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019111205/-/ 13. Levitan I, Volkov S, Subbaiah PV: Oxidized LDL: Diversity, patterns of rec- DCSupplemental. ognition, and pathophysiology. Antioxid Redox Signal 13: 39–75, 2010

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AFFILIATIONS

1CarMeN Laboratory, University of Lyon, Institut National de la Santé et de la Recherche Médicale (INSERM) U1060, Institut National des Sciences Appliquées de Lyon (INSA-Lyon), Claude Bernard University Lyon 1, Institut National de la Recherche Agronomique (INRA) U1397, Villeurbanne, France 2Department of Nephrology, University Hospital of Lyon, E. Herriot Hospital, Lyon, France 3Protein Science Facility, SFR BioSciences, Centre National de la Recherche Scientiﬁque (CNRS) UMS3444, INSERM US8, Claude Bernard University Lyon 1, École Normale Supérieure de Lyon (ENS de Lyon), Lyon, France 4Association Pour l’Utilisation du Rein Artiﬁciel dans la Région Lyonnaise (AURAL), Lyon, France 5Department of Endocrinology, University Hospital of Lyon, L. Pradel Hospital, Bron, France 6Institute for Molecular and Supramolecular Chemistry and Biochemistry, University of Lyon, INSA-Lyon, UMR 5246 CNRS, Villeurbanne, France

16 JASN JASN 31: ccc–ccc,2020 Supplementary materials

Supplementary Table of Content

Complete Methods

Supplementary Table 1: General characteristics of hemodialysis and control patients for aggregation assay

Supplementary Figure 1: Histological examinations of rabbit kidneys

Supplementary Figure 2: Examples of modified amino-acid spectra

Supplementary Figure 3: Spearman correlation of TxB2 assay and aggregation assay

Supplementary Figure 4: Spearman correlation of the amount of 4-HNE adducts on in-vitro modified HDL

Supplementary Figure 5: Immunobloting of 4-HNE adducts of HNE-modified HDL, HD, PD and Control HDL

Supplementary Figure 6: Aggregation levels of HNE-modified HDL vs control and CD36 blockade effect

Complete methods

Patients and ethics statement

Patients were sampled at the University Hospital of Lyon. Control patients were healthy volunteers for a living kidney donation, hospitalized for their pre-donation biological and clinical work-up. Hemodialysis patients were sampled within the hemodialysis unit of the Edouard Herriot hospital before the mid-week session. Inclusion criteria were age >18, undergoing hemodialysis for more than 6 months. Exclusion criteria were diabetes mellitus, ongoing inflammatory disease, liver cirrhosis, recent cardiovascular event (<3 months, cardiac myoinfarction, stroke, acute peripheral artery occlusion), uncontrolled anemia, coagulopathy and BMI higher than 35 kg.m-2. The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethical committee (reference L16-57, Comité de Protection des Personnes Lyon Sud Est IV). A written informed consent was obtained from all subjects.

Blood collection

Blood samples were obtained by venipuncture (on EDTA coated tubes) except for dialysis blood samples that were obtained immediately before dialysis from the arterial line of the mechanical bloodstream. Blood samples were centrifuged at 3500 x g for 10 min to isolate plasma which were collected and stored at -80°C until use.

Animal procedures

All animal experiments were performed under the authorization no. n°69-266-0501 and according to the guidelines laid down by the French Ministry of Agriculture (n° 2013-118) and the European Union Council Directive for the protection of animals used for scientific purposes of September 22, 2010 (2010/63UE). Adult male White New Zealand rabbits (CEGAVssc,

Saint Mars d'Egrenne, France) were housed in individual cages at constant ambient temperature

(21-23°C) and humidity (45-50%) with a 12 h light cycle. All animals had free access to tap

2 water. After a 7-day period of acclimation, rabbits were randomized to either the 5/6 nephrectomy group or in the control group.

Nephrectomy was performed as described by Goitloib et al.1 Rabbits were anaesthetized using an intramuscular injection of ketamine (50mg/kg), xylazine (5mg/kg), and acepromazine

(0.5mg/kg, Centravet, Lapalisse, France). The rabbit was placed on the right lateral decubitus.

The right dorsolumbar area was shaved and disinfected twice with povidone-iodine. A sterile field was applied to delimit the operative site and a sterile film was applied onto the shaved skin. Local anesthesia was performed by a subcutaneous injection of xylocaine 2% (2 ml) at the incision site. Then, a skin incision of approximately 4 cm in length was performed at 2 finger-breadths from the caudal end of the rib cage and from the lumbar muscle. The left kidney was externalized and the perirenal adipose tissue was gently dissected. The two poles of the kidney were electrocoagulated using an electric needle to produce a 2/3 reduction of the left kidney mass. The muscle and wound were sutured (Prolene 4/0, Ethicon, Somerville, NJ, USA), and the wound was painted with povidone-iodine. One week after surgery, a unilateral right nephrectomy was performed. A nylon monofilament ligature was placed on the kidney pedicle.

The kidney pedicle was carefully sectioned between the kidney and the ligature. Special care was taken to avoid damage to the adrenal glands. The control animals underwent the same procedures (general anesthesia, skin, and muscle incisions) followed by a simple kidney mobilization. All rabbits were given buprenorphine (0.05mg/kg sc, 2-times a day) for 2 days to prevent post-surgical pain. Food intake and body weight were measured on a daily and twice- weekly basis, respectively.

Measurement of glomerular filtration rate

The glomerular filtration rate (GFR) was measured through the kinetics of plasma iohexol concentration decrease. Rabbits were anesthetized with an intravenous injection of 27.5 mg/kg

3 of sodium pentobarbital (Centravet). Then, the neck was shaved and a median incision was performed after local anesthesia with a subcutaneous infiltration of xylocaine 2% (1mL). A

PE-50 catheter was introduced in the left jugular vein for the injections and PE-60 catheter was disposed in the left carotid artery for the blood sampling. A 1mL bolus of iohexol (Omnipaque

300®, GE Healthcare, Chicago, IL, USA) was performed and the timer was started after flushing the residue left in the jugular catheter with a saline solution. The syringe was weighted to the nearest milligram before and after injection to precisely calculate the amount of iohexol delivered to the rabbit. Blood was sampled at 5, 15, 30, 45, 60, 120, and 180 min on lithium- heparin coated tubes. The plasma was aliquoted after centrifugation at 5,900 x g for 2 min and stored at -20°C. The serum iohexol concentration was measured by HPLC as previously described2 and GFR was calculated using a bi-compartmental model equation.

Sacrifice and necropsy

At the end of GFR measurement, rabbits were deeply anesthetized with an overdose of sodium pentobarbital (70 mg/kg iv). Blood was removed by cardiac puncture and placed in EDTA- coated tubes. After a centrifugation at 1,250 x g for 10 min, plasma was aliquoted and stored at

-80°C. Urine were obtained with a direct bladder puncture and stored at -20°C. The kidney was dissected out, weighed, and stored in buffered formalin 10% (w/v) for histological examination.

Renal histology

Kidneys were dehydrated using ethanol, embeded in paraffin and sliced. Haematoxylin

Erythrosine Saffron (HES) and Sirius Red stainings were performed.

Isolation of lipoproteins from the plasma

Lipoproteins were separated from plasma by stepwise potassium bromide (KBr) density gradient ultracentrifugation.3 Plasma was fractionated in the Beckman ultracentrifuge with a rotor TAL 100.3 (Beckman, Brea, CA, USA). A first centrifugation at 100,000 x g for 3h 30min at 15°C was performed to remove the top layer corresponding to VLDL and chylomicrons.

Then, the plasma density was adjusted to 1.063 g.mL-1 with KBr (M=119.01 g.mol-1). After a second centrifugation at 100,000 x g for 5h at 4°C the orange ring, corresponding to LDL, was collected. Finally, plasma density was adjusted to 1.21 g.mL-1 with KBr and after centrifugation at 100,000 x g for 6 h 30 at 4°C the orange ring corresponding to HDL, was collected.

For the isolation of all the lipoproteins together, a single ultracentrifugation was performed at

100,000 x g for 6h 30min at 4° after an adjustment of plasma density to 1.21 g.mL-1 with KBr.

For platelet aggregation and copper-induced HDL oxidation assay freshly isolated HDL samples were used to prevent from HDL ultrastructure modification due to freezing,40 after the ultracentrifugation, lipoproteins were extensively dialyzed against phosphate saline buffer

(PBS) with 1mM EDTA for 3h, twice at room temperature and then overnight at 4°C. A last dialysis without EDTA was performed just before the platelet aggregation and copper-induced oxidation. HDL were stored at 4°C maximum 48 hours before the experiments.

Biochemistry

Serum creatinine measurement was performed using the Siemens enzymatic method (on the

Dimension Vista System, Siemens Healthcare, Erlangen, Germany) traceable to National

Institute of Standards and Technology Creatinine Standard Reference Materials 914 (verified with National Institute of Standards and Technology SRM 967) with calibration certified by isotope dilution mass spectrometry. Urea was measured with urease test (Vista 1500).

Cholesterol and triacylglycerols levels were measured using enzymatic kits (Biomerieux,

Marcy l’Etoile, France). HDL concentration was measured with an enzymatic kit (Abcam,

Paris, France). Malondialdehyde (MDA) was measured by HPLC coupled to UV-visible

5 detection (Diode Array detector) as described by Grotto et al.5. Antioxidant activity (AOA) of the plasma was measured as described by Koracevic et al.6. Proteinuria was measured using the

Bradford protein assay (BioRad, Marne-la-Coquette, France) using bovine serum albumin as a standard.

Lipoproteins assays

MDA concentration in HDL was determined by HPLC according to the method described by

Therasse and Lemonnier7. Lipoprotein samples, mixed with thiobarbituric acid (TBA, 10 mM), acetic acid and bultyl-hydroxy-toluen (5mM) were heated for 60min at 95°C. The TBA-MDA adducts were then extracted with ethyl acetate and separated on a Nucleosil C18 column (5μm,

4.6 × 250 mm, Macherey-Nagel, Hoerdt, France) using methanol/water (20:80, v/v) as mobile phase. Detection was performed by measuring the absorbance at 532 nm.

Anti-HNE-Michael adduct (ref 393207) and anti- HHE-Michael adduct (ref NOF-N213730-

EX) antibodies were purchased from Calbiochem (San Diego, CA, USA) and Cosmobio

(Tokyo, Japan), respectively. The antibody used to detect HHE-adducts is reported to be highly specific for HHE Michael adducts on histidine residues and therefore enables the specific detection of HHE-histidine in protein samples8. Fifty micrograms of HDL were loaded directly onto a nitrocellulose membrane using the Bio-Dot apparatus (BioRad, Marne-la-Coquette,

France). Following saturation with 5% bovine serum albumin (BSA), membranes were probed overnight with primary antibodies, anti-HHE-Michael adducts, or anti-HNE-Michael adducts

(1:1000 dilution in a 5% BSA solution). After incubation with HRP-coupled secondary antibodies (1:3000; 5% BSA solution), membranes were processed for chemiluminescence

(ECL plus, GE Healthcare, Chicago, IL, USA) and quantitated by densitometry using Image J software (NIH, Bethesda, MD, USA). The 8-isoprostane level was measured using an immunoassay (Bertin Pharma, Montigny-le-Bretonneux, France).

4-HNE was synthetized as described by Soulère et al.9 and was diluted in dimethylsulfoxyde

(DMSO). 4-HNE was added to HDL solutions at a final concentration of 1, 10, 50, or 100 µM.

After a 16h overnight incubation at 37°C in a water bath, HDL were dialyzed three times against

PBS to remove the free fraction of 4-HNE.

Platelet aggregation and activation

Blood was collected at the regional blood center from healthy volunteers who had not ingested any aspirin or any other non-steroidal anti-inflammatory drug in the previous 10 days. Platelets were prepared for the assays as described by Lagarde et al.10 Platelet function test was carried out according to the Born turbidimetric method.11 Platelet aggregation was measured in isolated platelets in a dual-channel aggregometer (Chrono-log; Coulter, Margency, France). Platelet suspensions were pre-incubated for 5 min at 37°C in the presence or absence of lipoproteins

(0.025 mg of protein/mL for rabbit, 0.050 mg/mL for human) and stimulated with threshold concentrations of collagen (75 ± 9 ng/ml) while being continuously stirred at 1,000 rpm. The threshold concentration of collagen was defined as the concentration of collagen that induced a

50% increase in light transmission. The extent of platelet aggregation was expressed in terms of percentage of change in light transmission 4 min after the addition of collagen. Blocking of

CD36 or SRB1 receptor was achieved by the pre-incubation with 10 µL of an anti-CD36 (Ab-

CD36) or anti-SRB1 (Ab-SRB1) antibodies (dilution 1:500, Abcam, Paris, France) for 10 min at 37°C before the incubation with HDL. Blocking of SRC-kinases was achieved by the preincubation with Naphtyl PP1 (final concentration 1 µM, Santa Cruz Biothechnologies, Dallas,

TE, USA) for 10 min at 37°C before the incubation with HDL. Aggregation values from lipoprotein assays were expressed as a percentage of the maximum aggregation induced by the collagen alone (considered as 100%).

For activation assays, platelet suspensions were pre-incubated for 15 min at 37°C in the presence or absence of lipoproteins (0.050 mg/mL for human) and stimulated with threshold concentrations of collagen (4ng/mL). Blocking of CD36 or SRB1 receptor was achieved by the pre-incubation with 10 µL of an anti-CD36 (Ab-CD36) or anti-SRB1 (Ab-SRB1) antibodies

(dilution 1:500, Abcam, Paris, France) for 10 min at 37°C before the incubation with HDL.

Blocking of SRC-kinases was achieved by the pre-incubation with Naphtyl PP1 (final concentration 1 µM, Santa Cruz Biothechnologies, Dallas, TE, USA) for 10 min at 37°C before the incubation with HDL. Levels of thromboxane B2 (TxB2) was measured using an immunoassay expressed as a percentage of the maximum level induced by the collagen alone

(considered as 100%, Cayman Chemical, Ann Arbor, MI, USA). After pre-incubation with

HDL and antibodies or pharmacological inhibitor, platelets were activated with collagen then fixed in paraformaldehyde 5% w/v and stained with anti-CD62/P-Selectin (Thermo Fisher

Scientific, San Jose, CA, USA) according to the manufacturer protocol and analyzed by flow cytometry.12 Expression of intra-platelet phosphorylated JNK was measured by Western Blot with an anti-phospho-JNK antibody (Cell signaling technologies, Danvers, MA, USA).

Quantification and expression were processed as previously described.13

Copper-induced HDL oxidation

Aliquots of freshly dialyzed HDL (50 µg protein) were oxidized in the presence of 2.5 µM of

CuSO4 within the day following the last dialysis. The oxidation was monitored by measuring the formation of conjugated dienes at 234 nm14 every 5 min for 3 h in a Kontron computer- linked spectrophotometer.

Mass spectrometry assay

List of reagents:

Sigma Aldrich (Saint Quentin Fallavier, France): Acetonitrile MS grade (ACN), Formic acid

(FA), Trifluoroacetic acid (TFA), Iodoacetamide (IAA), Dithiotreitol (DTT), Sodium

Deoxycholate (SDC), Ammonium Bicarbonate (AB)

MilliQ Water

Trypsin: Promega (Lyon, France)

Pierce C18 desalting spin column and Pierce quantitative fluorometric peptide assay: Thermo

Scientific (San Jose, CA, USA)

In-solution digestion

30 µg of protein were diluted in 100 µL of 50 mM AB/SDC 1% buffer. They were reduced with 5 mM DTT for 1h at 57°C, and then alkylated with 10 mM IAA for 1h in the dark at room temperature and under agitation (850 rpm). Enzymatic digestion was performed with trypsin at a ratio 1/100 (enzyme/proteins) and overnight at 37°C. Endly, the samples were desalted on

C18 spin column and dried on Speed-Vacuum before nanoLC-MS/MS analysis.

LC-MS/MS analysis

Prior to injection, the peptides concentrations in the samples were determined by the Pierce quantitative fluorometric peptide assay (Thermo scientific) according to the manufacturer’s instructions. An aliquot containing 1µg for each sample was dried and suspended in 9 µL of

FA 0.1% + 1 µL of Cytochrome C digest (2 pmol/µL) used as internal standard. 2µL of this solution was then injected. Samples were analyzed in a Label Free quantitation strategy, in triplicate using an Ultimate 3000 nano-RSLC (Thermo Scientific) coupled on line with a Q

Exactive HF mass spectrometer via a nano-electrospray ionization source (Thermo Scientific,

San Jose California).

Nano-LC: Samples were injected and loaded on a C18 Acclaim PepMap100 trap-column 75

µm ID x 2 cm, 3 µm, 100Å, (Thermo Scientific) for 3.0 minutes at 5 µL/min with 2% ACN,

0.05% TFA in H2O and then separated on a C18 Acclaim Pepmap100 nano-column, 50 cm x

75 m i.d, 2 µm, 100 Å (Thermo Scientific) with a 60 minutes linear gradient from 4% to 50% buffer B (A: 0.1% FA in H2O, B: 100% ACN, 0.1% FA) and then from 50 to 95% of B in 2 min, hold for 10 min and returned to the initial conditions in 1 min for 14 min. The total duration was set to 90 minutes at a flow rate of 300 nL/min. The oven temperature was kept constant at

40°C.

MS: Samples were analysed with a TOP20 HCD method: MS data were acquired in a data dependent strategy selecting the fragmentation events based on the 20 most abundant precursor ions in the survey scan (300-1600 Th). The resolution of the survey scan was 60,000 at m/z 200

Th and for MS/MS scan the resolution was set to 15,000 at m/z 200 Th. The Ion Target Value for the survey scans in the Orbitrap and the MS/MS scan were set to 3E6 and 1E5 respectively and the maximum injection time was set to 60 ms for MS scan and for MS/MS scan. Parameters for acquiring HCD MS/MS spectra were as follows; collision energy = 27 and an isolation width of 2.0 m/z. The precursors with unknown charge state, charge state of 1 and 8 or greater than 8 were excluded. Peptides selected for MS/MS acquisition were then placed on an exclusion list for 20 s using the dynamic exclusion mode to limit duplicate spectra.

Data Analysis

Proteins were identified by database searching using SequestHT with Proteome Discoverer 2.2 software (Thermo Scientific) against the Swissprot Homo Sapiens database (2018-07 release,

24480 sequences). Precursor mass tolerance was set at 10 ppm and fragment mass tolerance was set at 0.02 Da, and up to 2 missed cleavages were allowed. Oxidation (M), acetylation

(Protein N-terminus) and HNE (+ 156.115 Da on K or H) were set as variable modifications, and Carbamidomethylation (C) as fixed modification. Peptides and proteins were filtered with a false discovery rate (FDR) at 1% using percolator and proteins were identified with 1 unique peptide in rank 1. Protein quantitation was performed with Minora feature detector and

10 precursor ions quantifier node in Proteome Discoverer 2.2 software with protein quantitation based on pairwise ratios and ANOVA (individual proteins) hypothesis test.

The mass spectrometry proteomics data have been deposited to the ProteomeX change

Consortium via the PRIDE partner repository with the dataset identifier PXD013301 (DOI:

10.6019/PXD013301; username: [email protected]; password: oMD4gyDT).

Molecular modelling

To study the accessibility of residues, the structure of truncated human apolipoprotein A-I (pdb code 1av1) was downloaded from the PDB database. All observations were performed using

PyMOL as software by representing the α-helices and histidine and lysine residues (colored in blue) in CPK to facilitate the study. The same method was achieved with the HDL model which was download from the PDB database.

Statistical analysis

Data were expressed as median and interquartile range. All analyses were performed using

GraphPad Prism version 6.0 (GraphPad software, La Jolla, CA, USA). Normality was assessed using D’Agostino & Pearson test. Comparisons were performed using Mann-Whitney U-test.

Differences were considered as significant at the P<0.05 level.

References

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2. Florens N, Lemoine S, Pelletier CC, Rabeyrin M, Juillard L, Soulage CO: Adenine Rich Diet Is Not a Surrogate of 5/6 Nephrectomy in Rabbits. Nephron 135: 307–314, 2017

3. Havel RJ, Eder HA, bragdon JH: The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J. Clin. Invest. 34: 1345– 1353, 1955

4. Holzer M, Kern S, Trieb M, Trakaki A, Marsche G: HDL structure and function is profoundly affected when stored frozen in the absence of cryoprotectants. J. Lipid Res. 58: 2220–2228, 2017

5. Grotto D, Santa Maria LD, Boeira S, Valentini J, Charão MF, Moro AM, Nascimento PC, Pomblum VJ, Garcia SC: Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection. Journal of Pharmaceutical and Biomedical Analysis 43: 619–624, 2007

6. Koracevic D, Koracevic G, Djordjevic V, Andrejevic S, Cosic V: Method for the measurement of antioxidant activity in human fluids. Journal of Clinical Pathology 54: 356–361, 2001

7. Therasse J, Lemonnier F: Determination of plasma lipoperoxides by high-performance liquid chromatography. J Chromatogr 23: 237-41, 1987

8. Yamada S, Funada T, Shibata N, Kobayashi M, Kawai Y, Tatsuda E, Furuhata A, Uchida K: Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids. J. Lipid Res. 45: 626–634, 2004

9. Soulère L, Queneau Y, Doutheau A: An expeditious synthesis of 4-hydroxy-2E- nonenal (4-HNE), its dimethyl acetal and of related compounds. Chemistry and Physics of Lipids 150: 239–243, 2007

10. Lagarde M, Bryon PA, Guichardant M, Dechavanne M: A simple and efficient method for platelet isolation from their plasma. Thromb. Res. 17: 581–588, 1980

11. BORN GV: Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194: 927–929, 1962

12. Skinner SC, Diaw M, Pialoux V, Mbaye MN, Mury P, Lopez P, Bousquet D, Gueye F, Diedhiou D, Joly P, Renoux C, Sow D, Diop S, Ranque B, Vinet A, Samb A, Guillot N, Connes P: Increased Prevalence of Type 2 Diabetes-Related Complications in Combined Type 2 Diabetes and Sickle Cell Trait. Diabetes Care 41: 2595–2602, 2018

13. Lê QH, Alaoui El M, Véricel E, Ségrestin B, Soulère L, Guichardant M, Lagarde M, Moulin P, Calzada C: Glycoxidized HDL, HDL enriched with oxidized phospholipids and HDL from diabetic patients inhibit platelet function. The Journal of Clinical Endocrinology & Metabolism 100: 2006–2014, 2015

14. Esterbauer H, Gebicki J, Puhl H, Jürgens G: The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13: 341–390, 1992

Supplementary Table 1. General characteristics of hemodialysis and control patients for aggregation assay Control Hemodialysis (HD) P-value N 9 9 General characteristics Age, years 41.78 ± 4.50 57.11 ± 5.60 0.049 Gender, % male 66.67 ± 16.67 55.56 ± 17.57 0.653 BMI, kg/m2 25.42 ± 1.06 22.89 ± 1.43 0.186 Comorbidities HT, n 2 8 Stroke, n 0 0 CHD, n 0 2 Cardiopathy, n 0 4 PVD, n 0 1 Therapies Statins, n 0 6 PI, n 0 5 RASi, n 1 3 ß-blockers, n 1 5 CCB, n 1 1 Biological parameters Urea, mmol/L 6.20 ± 0.51 15.00 ± 1.62 <0.0001 Creatinine, µmol/L 78.78 ± 4.70 612.10 ± 52.95 <0.0001 mGFR, mL/min/1.73m2 92.33 ± 4.08 - Total cholesterol, mg/dL 211 ± 9 153 ± 18 0.011 LDL cholesterol, mg/dL 132 ± 8 87 ± 16 0.025 HDL cholesterol, mg/dL 58 ± 2 44 ± 3 0.005 Triacylglycerols, mg/dL 105 ± 10 109 ± 9 0.749 CRP, mg/L 3.00 ± 1.13 18.83 ± 11.29 0.200 Data are expressed as mean ± SEM. BMI: body mass index; HT: hypertension; CHD: coronary heart disease; PVD: peripheral vascular disease; PI: platelet inhibitor; RASi: renin-angiotensin system inhibitor; CCB: calcium-channel blocker; eGFR: estimated glomerular filtration rate by CKD-EPI formula; mGFR: measured GFR by iohexol clearance; CRP: C-reactive protein. Creatinine: x 0.011 for mg/dl; urea: x 2.8 for mg/dl

Supplementary Figure 1

Figure 1: Histological examinations of rabbit kidneys with hematoxylin phloxine saffron (HPS) staining. A: The electrocoagulated cortex was replaced by a fibrous layer (black arrow) and some glomeruli were surrounded by fibrosis. (x2.5) B: Remnant glomeruli exhibiting hypertrophy (black arrow; x10). Mean glomerular radius was 23 in control group vs 29 in CKD group (p<0.05) C: Peritubular fibrous involution (black arrow) (x10) D: Note the large number of of glomeruli and the fibrosis restricted to perivascular areas (black arrow) (x2,5) E — F: Glomeruli were smaller (black arrow) than in CKD group and fibrosis was restricted to perivascular areas (x10).

Supplementary Figure 2

K250: A: unmodified peptide; B: modified peptide X:\permanent\...\180830-NF0607-03 08/31/18 18:28:10 ech 607 H6 patient HDL 100 ng/uL+ CytC 200 fmol/uL,2ulPeptide inj col 50cm, [241 grad-250] 60min : MH+ : 1230.7110 Th - MSMS on m/z 615.8591 (2+ ; +1.41 ppm) 180830-NF0607-03 #26550 RT: 48.10 AV: 1 NL: 2.70E5 T: FTMS + c NSI d Full ms2 [email protected] [100.0000-1275.0000] 819.4597 100 y + 95 7

90 b2 b3 b4 A 85 Q-G-L-L-P-V-L-E-S-F-K y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 80

65 + b3 60 299.1711 55

RelativeAbundance 40

30 + b2 25 120.0809 186.0873 + + 281.1609 y3 y 168.0766 312.1526 606.8516 + 8 20 y5 381.2132 y 2+ + 7 y4 932.5446 15 y + y + 1 510.2538 6 197.1283 y + + 10 + 526.2896 722.4088 9 y10 y2 366.2480 832.4901 217.7114 439.2556 1045.6222 5 + 492.2427 778.4208 1102.6440 b4 590.3613 655.3385 919.5132 545.6111 1003.5416 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 m/z X:\permanent\...\180828-NF0601-03 08/29/18 08:47:17 + + ech 601 C10 patient controle 100 ng/uL+Peptide CytC 200 [241 fmol/uL,2ul-250] inj col HNE 50cm, gradModification 60min : MH : 1386.8163 Th - MSMS on m/z 462.9436 (3 ; +5.79 ppm) 180828-NF0601-03 #20002 RT: 38.06 AV: 1 NL: 1.36E5 T: FTMS + c NSI d Full ms2 [email protected] [100.0000-1440.0000] 779.4417 100 + b b b b b b y5 95 1 2 3 4 5 6 B 2+ Q-G-L-L-P-V-L-E-S-F-KHNE y7 90 y8 y7 y6 y5 y4 y3 y2 y1 488.2854 85 169.1339 + y4 80 666.3578 75

65 + y3 60 394.2456 537.3144 55 197.1289

50 136.0760 360.0290 45

RelativeAbundance 40 520.2858 651.3466 35

30 249.1597 590.3677 430.0891 3+ 25 y7 + y6 2+ + y3 325.8596 y7 20 b + 878.5141 269.1612 3 2+ 975.5671 217.8265 y 2+ y6 y + y 2+ 15 299.1725 5 2 8 + + b4 456.9375 10 + y1 b2 764.4250 + 562.3692 + 703.4490 b6 5 b5 608.3729 931.1113 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 m/z

H179: A: unmodified peptide; B: modified peptide X:\permanent\...\C8-180830-NF598-01 08/30/18 20:17:28 ech 598 C8 patient controle 100 ng/uL+ CytC 200 fmol/uL,2ulPeptide inj col [17850cm, grad-184] 60min : MH+ : 781.4313 Th - MSMS on m/z 391.2193 (2+ ; -0.24 ppm) C8-180830-NF598-01 #6158 RT: 18.42 AV: 1 NL: 8.31E5 T: FTMS + c NSI d Full ms2 [email protected] [100.0000-820.0000] 209.1032 100 + 95 b2

90 b2 b3 b4 A + y5 85 A-H-V-D-A-L-R y6 y5 y4 y3 y2 y1 573.3345 80

50 + y4 45 + 149.0232 y3 474.2666 RelativeAbundance 40 181.1083 359.2395 110.0716 2+ 35 y6 30

+ 25 y1 20 + b3 15 136.0868 308.1712 10 237.1342 391.2204 + + 286.1393 b4 y6 5 + y2 457.2414 556.3179 342.2118 423.1978 494.2357 583.3207 710.3953 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z X:\permanent\...\180831-NF0595-02 09/03/18 16:34:53 ech 595 C4 patient controle 100 ng/uL+Peptide CytC 200 fmol/uL,2ul [178-184] inj col 50cm, HNE grad modification 60min : MH+ : 937.5467 Th - MSMS on m/z 313.1871 (3+ ; +0.13 ppm) 180831-NF0595-02 #11650 RT: 25.66 AV: 1 NL: 4.52E4 T: FTMS + c NSI d Full ms2 [email protected] [100.0000-980.0000] 175.1190 100 + B y1 95 b2 b3 b4 90 A-HHNE-V-D-A-L-R 85 + y2 y5 y4 y3 y2 y1 80 288.2026

70 139.1116 65 + b2 60 365.2179

50 202.1075

45 218.5073

RelativeAbundance + 40 y3 35

30 + b4 257.2473 25 579.3127

15 + + y + b3 y4 5 313.2143 10 667.3018 464.2864 561.3123 5 416.2299 492.0358 802.0598

0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

H189: A: unmodified peptide; B: modified peptide X:\permanent\...\180828-NF0599-03 08/28/18 20:03:04 + + ech 599 C7 patient controle 100 ng/uL+ CytC 200 fmol/uL,2ulPeptide inj col [185 50cm,- grad195] 60min : MH : 1301.6486 Th - MSMS on m/z 651.3279 (2 ; +0.13 ppm) 180828-NF0599-03 #10536 RT: 24.32 AV: 1 NL: 2.47E5 T: FTMS + c NSI d Full ms2 [email protected] [100.0000-1350.0000] 239.1139 100 + b2 95 b2 b3 b4 90 T-H-L-A-P-Y-S-D-E-L-R 85 y10 y9 y8 y7 y6 y5 y4 y3 y2 y1 A 80 + 75 y7

70 879.4197 + 65 b3 352.1979 + 60 y8

55 950.4571 + y9 50 1063.5410 45

RelativeAbundance 40

25 + 195.0879 b4 20 424.7045 + y1 + 15 y2 + 175.1192 334.1863 y3 288.2044 651.8331 10 392.7036 579.8117 + + y4 y6 860.4546 5 848.3995 973.5197 463.1810 532.2737 782.3676 932.4443 1158.6031 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z X:\permanent\...\180828-NF0602-02 08/29/18 13:36:52 ech 602 H1 patient HDL 100 ng/uL+Peptide CytC 200 fmol/uL,2ul [185- inj195] col 50cm, HNE grad Modification 60min : MH+ : 1457.7636 Th - MSMS on m/z 486.5927 (2+ ; +0.08 ppm) 180828-NF0602-02 #16712 RT: 33.31 AV: 1 NL: 2.09E5 T: FTMS + c NSI d Full ms2 [email protected] [100.0000-1510.0000] 579.3503 100 + b4 95 b1 b2 b3 b4 b5 b6 B 90 T-HHNE-L-A-P-Y-S-D-E-L-R 85 y7 y6 y5 y4 y3 y2 y1 + 80 y5 75 619.3040

RelativeAbundance + 40 y2 y + + 4 35 288.2031 y3 532.2719 417.2455 30 + 25 b3 + 508.3120 y1 + 20 b2 136.0759 175.1189 + 395.2287 y 338.7038 486.3055 6 15 + 782.3687 b6 10 203.0661 839.4709 233.1284 431.2089 b + 324.7069 670.3382 5 764.3541 + 5 271.1761 551.3569 y7 629.2820 382.2108 440.2132 697.3928 744.8077 811.4622 861.4108 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

Supplementary Figure 3

Supplementary Figure 2

100

% aggregation 20

0 0 50 100 150 200 250 % activation (TxB2)

Spearman correlation of TxB2 assay and aggregation assay of 7 control patients and 8 hemodialysis patients. r2=0.3894, p=0.0129

Supplementary Figure 4 Supplementary Figure 2

3.5

3.0

2.5 AU 2.0

1.5

1.0 0 10 50 100 HNE uM

Sperman correlation of the amount of 4-HNE adducts on HDL from in vitro adduction. Significant correlation was observerd (p=0.0179)

Supplementary Figure 5

4-HNE adducts are increased in HDL from hemodialysis (HD), peritoneal dialysis (PD) patients and 4- HNE in vitro modified HDL (HNE) compared to controls (CTL). No difference were however observed in the level of adduction between HD, PD and HNE HDL. Immunoblotting (dot blot) of 4-hydroxynonenal (4-HNE) Michael adducts were performed as described in methods section. n=5 for each group *, p<0.05, Mann-Whitney U test vs HD, PD and HNE HDL

Supplementary Figure 5 Supplementary Figure 6

* * 250 *

200

150

100

% of aggregation 50

0 Ab Anti-CD36 - + - + - +

Collagen Collagen Collagen + + HV Control HNE HDL HDL

Healthy volunteers (HV) HDL modified by an incubation overnight with HNE (HNE HDL) solution exhibited pro-aggregant properties compared to non-modified HDL (HV Control HDL, C). Pre- incubation with anti-CD36 antibody restored HNE HDL anti-aggregant properties like HV Control HDL (n=4 for each group). *p<0.05