Dialysis Modalities and HDL Composition and Function

Abstract

Lipid abnormalities may have an effect on clinical outcomes of patients on dialysis. Recent studies have indicated that HDL dysfunction is a hallmark of ESRD. In this study, we compared HDL composition and metrics of HDL functionality in patients undergoing hemodialysis (HD) or peritoneal dialysis (PD) with those in healthy controls. We detected a marked suppression of several metrics of HDL functionality in patients on HD or PD. Compositional analysis revealed that HDL from both dialysis groups shifted toward a more proinflammatory phenotype with profound alterations in the lipid moiety and protein composition. With regard to function, cholesterol efflux and anti-inflammatory and antiapoptotic functions seemed to be more severely suppressed in patients on HD, whereas HDL-associated paraoxonase activity was lowest in patients on PD. Quantification of enzyme activities involved in HDL metabolism suggested that HDL particle maturation and remodeling are altered in patients on HD or PD. In summary, our study provides mechanistic insights into the formation of dysfunctional HDL in patients with ESRD who are on HD or PD.

Keywords: cardiovascular disease; hemodialysis; lipids; peritoneal dialysis.

Figure 1.

HD HDL shifts toward the smaller HDL3 subclass. HDL (5 µg protein/lane) was separated by native gradient gel electrophoresis and stained with Coomassie Brilliant Blue. Intensity blots of individual samples were obtained, and the peak areas of HDL₂ and HDL₃ were calculated. Total peak area was set to 100% and used to calculate the percentage of HDL₂/HDL₃. Values shown represent means±SEMs of three individual experiments. CON, control.

Figure 2.

Cholesterol efflux capability is impaired in HDL from patients on HD or PD. (A) HDL levels from 24 patients on HD, 14 patients on PD, and 20 control subjects (CON) were examined for their ability to efflux [³H]-cholesterol from TO-901317–stimulated, lipid-loaded RAW 264.7 macrophages. [³H]-cholesterol–labeled cells were incubated with HDL (50 µg/ml) for 2 hours at 37°C. Cholesterol efflux is expressed as the radioactivity in the medium relative to total radioactivity in medium and cells. (B) HDL was labeled with DiI and examined for its ability to promote lipid uptake by HepG2 cells. Cellular uptake of the fluorescent lipophilic dye DiI was quantified by flow cytometry. Values shown represent means of three independent experiments.

Figure 3.

Functional analyses reveal dysfunctional HDL in HD and PD patients. (A) Antioxidative activity of HDL was measured by inhibition of free radical-induced oxidation of DHR. Increase in fluorescence was monitored over time at 538 nm, and inhibition was calculated from slopes of individual samples. Results are expressed as percentage of inhibition compared with DHR oxidation in the absence of HDL. (B) Anti-inflammatory function of HDL was tested using a human monocyte cell line containing a reporter cassette for the NF-κB, which induces GFP expression on NF-κB translocation into the nucleus. Cells were pretreated for 90 minutes with 50 µg/ml HDL and stimulated for 24 hours with 50 ng/ml LPS. Afterward, GFP expression was assessed by flow cytometry. (C) Arylesterase activity of HDL-associated paraoxonase was measured by using phenylacetate as substrate. (D) Lp-PLA₂ activity of HDL was measured using 2-thio platelet activating factor as substrate. The arylesterase and Lp-PLA₂ activities of HDL were calculated from the slopes of the kinetic chart of three independent experiments. (E) Human coronary artery endothelial cells were incubated in serum-containing (+SERUM) or serum-free (−SERUM) media in the absence or presence of 150 µg/ml HDL from healthy controls (CON-HDL), patients on HD (HD-HDL), or patients on PD (PD-HDL) overnight. Subsequently, cells were stained for caspase 3/7 and imaged by confocal microscopy. (A–D) All values shown represent means of two independent experiments. CON, control.

Figure 4.

Analysis of serum enzymes indicates that HDL metabolism is impaired in dialysis patients. Serum samples from 24 patients on HD, 14 patients on PD, and 20 control subjects (CON) were examined for activities of (A) LCAT, (B) PLTP, (C) CETP, (D) SN1 lipases, and (E) SN2 lipases. Values shown represent means of two independent experiments.

Figure 5.

Dialysis modalities and HDL composition and function. Shown are alterations in protein content on the outer circle, where dark red-colored proteins indicate proteins that are highly remodeled (>50%) and light red-colored proteins indicate moderately altered proteins (<50%). Functional alterations of HDL as well as modulation of serum enzyme activities are indicated with arrows. ↓↓ and ↑↑ indicate highly altered functions, and ↓ and ↑ indicate moderately altered functions. E, apolipoprotein E; PON, paraoxonase.