BASIC RESEARCH www.jasn.org CKD Increases Carbonylation of HDL and Is Associated with Impaired Antiaggregant Properties 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 cardio- vascular events. Modifications induced by increased oxidative stress particularly affect circulating lipo- proteins 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 acti- vation 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 cardiovas- cular 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 JASN 31: ccc–ccc, 2020 ISSN : 1046-6673/3107-ccc 1 BASIC RESEARCH www.jasn.org 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 ne- phrectomy 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