Patients with chronic kidney disease (CKD) are at high risk for cardiovascular disease (CVD). However, traditional lipid risk factors, including low HDL levels, cannot completely explain the increased risk. Altered HDL proteome is linked with both CVD and CKD, but the role of HDL proteins in incident CVD events in CKD is unknown. In this prospective case-control study, we used targeted proteomics to quantify 31 proteins in HDL from 92 subjects (46 incident new CVD and 46 one-to-one matched controls) at various stages of CKD. We tested associations of HDL proteins with incident CVD using matched logistic regression analysis. In unadjusted models, levels of six HDL proteins (APOA1, APOA4, APOC3, LCAT, PON1, and PON3) significantly associated with incident CVD. No significant associations were found for HDL-C. In the fully adjusted model for clinical confounders and lipid levels, we observed an inverse association between levels of PON1, PON3, and LCAT in HDL and incident CVD. Odds ratios (per 1-SD) were 0.38 (0.18-0.97, P=0.042), 0.42 (0.20-0.92, P=0.031), and 0.30 (0.11-0.83, P=0.020) for PON1, PON3, and LCAT, respectively. APOA4 remained associated with incident CVD in CKD patients in models adjusted for clinical confounders and lipid levels but lost significance with the addition of CRP and proteinuria in the model. In conclusion, levels of four HDL proteins, PON1, PON3, LCAT, and APOA4, were inversely associated with incident CVD events in CKD subjects. Our observations indicate that HDL’s protein cargo but not HDL-C levels can serve as a marker—and perhaps mediator—for elevated CVD risk in CKD patients.

To quantitatively measure the relative levels of HDL proteins, we used targeted proteomics with isotope-dilution parallel reaction monitoring (PRM). PRM analyses were performed in the positive ion mode with an ultrahigh-resolution accurate mass Orbitrap Fusion Tribrid Mass Spectrometer coupled to a nanoACQUITY UPLC. Initially, the potential peptides for each protein were selected from the detected peptides by shotgun analysis and from our previous studies. At least two peptides from one protein were then tested by PRM test runs and finally 2 or more peptides were selected for 21 proteins, and 1 peptide for 10 proteins (Supplemental Table 1). The targeted PRM MS data of peptides of each HDL protein were analyzed using Skyline. An equal amount of 15N-labeled APOA1 was added to HDL isolated from each subject prior to digestion as an internal standard. The peak areas of all the transitions of a peptide detected by PRM analysis were summed to get the total peak area for the peptide but the transitions with interferences were deleted. To normalize the peak area of a peptide, the total peak area of all selected transitions of the peptide was divided by the peak area of each of the four 15N-labeled peptides from 15N-APOA1 and the ratios were used for quantification. To calculate the relative levels of the peptide between control and incident CVD groups, we set each of the average ratio of the peptide in control subjects as an arbitrary unit of one and the average of the four ratios was set as the relative level of the peptide. If two or more peptides were quantified for a protein, the relative levels of all peptides from the protein were averaged to obtain the relative level of that protein in HDL.

We used plasma samples from the CPROBE cohort that were collected at the time of enrolment. The 46 cases were individually matched to a control subject of the same sex and diabetic status in a one-to-one ratio. HDL (density 1.063-1.210 g/mL) was isolated by sequential ultracentrifugation from rapidly thawed plasma. 5 µg of HDL proteins and 0.2 µg of isotope-labeled [15N]APOA1 (added as the internal standard) were digested by trypsin. And the digested peptides were analyzed to determine the relative levels of 31 proteins in HDL.