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

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 µM of HNE.

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.

Keywords: cardiovascular disease; chronic kidney disease; dialysis; dyslipidemia; platelets; thrombosis.

Graphical abstract

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. n=8 and 9, CKD and control, respectively; *P<0.05, Mann–Whitney test. Data are expressed as median and interquartile range.

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. Collagen 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. n=8 and 9, CKD and control, respectively. *P<0.05, Mann–Whitney test. Data are expressed as median and interquartile range.

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) 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). n=9 and 9, HD and control HDL, respectively. * P<0.05, Mann–Whitney test. Data are expressed as median and interquartile range.

Figure 4.

Structure of truncated human apo A-I (pdb code 1av1) revealed several accessible sites for post-translational modifications by 4-HNE. Four molecules are associated with their hydrophobic faces to form an antiparallel four-helix bundle. (A) Lysine and histidine residues possibly modified by HNE are colored in cyan with CPK representation. (B) Model of HDL:phospholipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) are colored in yellow and cholesterol in magenta. Lysine and histidine residues possibly modified by HNE are colored in cyan with CPK coloring representation (pdb code 3k2s).

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 (n=15 for control, n=25 for HD group, and n=20 for PD group). (B) The same observations were found for healthy volunteers’ HDL modified by an incubation overnight with HNE (HNE HDL, n=5 for each group). (C) HD, PD, and HNE HDL exhibited blunted antiaggregant properties compared with control HDL (n=9 for control and HD groups, n=5 for HNE group). (D) We found a threshold effect of HNE modification on HDL because only a 100 µM solution of HNE permitted the significant alteration of the antiaggregant properties of control HDL (n=4 for each group). *P<0.05, Mann–Whitney test and Kruskal–Wallis test (in D). Data are expressed as median and interquartile range.

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 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. n=5 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.

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.