Subjects with DM2 with DKD were recruited at the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. The inclusion criteria were based on the diagnosis of DM2 for at least 10 years and on the classification of chronic kidney disease according to the loss in the glomerular filtration rate (eGFR; < 60 mL/min/1.73 m2) and enhanced urinary albumin excretion rate [AER; A1: < 30 mg/g creatinine; A2: 30 - 300 mg/g creatinine and A3: > 300 mg/g creatinine] for at least 3 months . HbA1c values were between 7 and 10%, avoiding greater variations in DM control and carbonyl stress due to hyperglycemia.
Healthy control individuals matched by age were selected at Faculdade de Medicina da Universidade de São Paulo. All participants were properly informed about the procedures and the study and signed an informed written consent form that was previously approved by The Ethical Committee for Human Research Protocols of the Clinical Hospital (#15024), in accordance with the Declaration of Helsinki. Subjects on dialysis, with other chronic diseases, rapid loss in eGFR (> 3 mL/min/year), refractory hypertension, BMI < 18.5 kg/m2, current smokers or those who abused alcohol were not included. Eighty percent of subjects with DKD were using insulin, and 70% were on statins and beta-blockers. Angiotensin-converting-enzyme inhibitors (ACEi) and metformin were used for A1 and A2 subjects only, and angiotensin II receptor blockers (ARBs) were used by groups A1 (39%), A2 (50%) and A3 (36%). Erythropoietin was utilized by the A2 (7%) and A3 (12%) groups.
Blood was drawn after overnight fasting, and HbA1c was determined by high-performance liquid chromatography (HPLC). Plasma was immediately isolated from the same sample in a refrigerated centrifuge (4°C). Glycemia, triglycerides (TG), total cholesterol (TC), HDL cholesterol (HDLc), fructosamine, TSH, creatinine and urea were determined in plasma by enzymatic techniques after overnight fasting and albumin in 24 h urine. Individuals with DKD were categorized according to eGFR above 60 mL/min/1.73 m2 plus AER stages A1 (< 30 mg/g creatinine) and A2 (30 - 300 mg/g creatinine) and eGFR below 60 mL/min/1.73 m2 plus stage A3 (> 300 mg/g creatinine). Control subjects presented eGFR above 60 mL/min/1.73 m2 plus A1.
Isolation of lipoproteins
Venous blood samples were drawn after overnight fasting, and plasma was immediately isolated in a refrigerated centrifuge. Preservatives were added to the plasma, and the density was adjusted with bromide potassium to 1.21 g/mL. Low-density (LDL; d = 1.019-1.063 g/mL) and high-density lipoprotein (HDL; d = 1.063-1.21 g/mL) were isolated from plasma by discontinuous density gradient ultracentrifugation (100,000g, 24 h, 4°C, Sw40 rotor; Beckman ultracentrifuge). Samples were dialyzed against phosphate-buffered saline containing EDTA (PBS).
HDL composition in lipids and apoA- I
The amount of lipids and apoA-I in HDL were determined by enzymatic techniques [TC and TG; Labtest diagnóstica S. A., Minas Gerais, Brazil; phospholipids; Randox Laboratorier LTD. Crumlin, Co., Antrem, United Kingdom] and immunoturbidimetry.
Determination of total AGEs and pentosidine in HDL
The contents of total AGEs and pentosidine were determined in isolated HDL by fluorescence measurement (Synergy HT Multi-Mode Microplate Reader, SpectraMax M5). Samples were excited at a wavelength of 370 nm, and the fluorescence emitted was 440 nm and 378 nm for total AGEs and pentosidine, respectively .
Determination of HDL carbamoylation
HDL carbamoylation was determined in the isolated lipoprotein by ELISA (- STA-877 Protein Carbamylation Sandwich ELISA; Cell Biolabs Inc., San Diego, CA, USA).
Determination of carboxymethyllysine in plasma
Carboxymethyllysine (CML) was determined in plasma by ELISA (Circulex CML, Woburn, MA, USA).
Proteolytic digestion of HDL
The HDL protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). Ten micrograms of HDL protein was solubilized in 100 mM ammonium bicarbonate, dithiothreitol, and iodoacetamide, following digestion with trypsin (1:40, w/w Promega, Madison, WI, USA) for 4 h at 37°C. Trypsin was further added to the samples (1:50, w/w HDL), and incubation was performed overnight at 37°C. Samples were desalted using solid phase extraction (Oasis PRIME HLB SPE column; Waters) after acidic hydrolysis with 2% trifluoroacetic acid, dried and kept frozen at -80°C until MS analyses. Prior to MS analysis, samples were resuspended in 0.1% formic acid (final protein concentration of 25 ng/μL).
Angiotensin peptide (DRVYIHPFHL, 0.2 pmol/µL) spiked in each sample was used as a global internal standard to control the robustness of the PRM methodology. Variability in the integrated peptide area was monitored across 87 injections, and low variance was obtained with a CV of 13%.
Targeted proteomic analyses
Digested HDL proteins (50 ng protein) were quantified by parallel reaction monitoring (PRM), as previously described . Briefly, an Easy-nLC 1200 UHPLC (Thermo Scientific, Bremen, Germany) was used for peptide separation. Each sample was loaded onto a trap column (nanoViper C18, 3 µm, 75 µm × 2 cm, Thermo Scientiﬁc), and after, the trapped peptides were eluted onto a C18 column (nanoViper C18, 2 µm, 75 µm × 15 cm, Thermo Scientiﬁc). Acquisition of the data was performed in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, Bremen, Germany) using a nanospray Flex NG ion source (Thermo Scientific, Bremen, Germany). A scheduled (3-min window) inclusion list containing m/z of precursor peptides of interest and corresponding retention times was generated using Skyline software .
Selection of HDL peptides for targeted quantification
PRM methodology was assembled using data derived from shotgun proteomics analyses as previously described . Ninety-one proteins were identified, but this number was reduced to 47 proteins after eliminating proteins that could be potential contaminants or were in low abundance (keratin, proteins with <2 unique peptides and peptides with high interfering signal). Peptides susceptible to ex vivo modification (e.g., methionine-containing peptides) were also avoided, and only peptides satisfactorily detected (with a good chromatographic peak, containing at least 4 coeluted transitions, and with mass error <10 ppm) were included in the final analysis. After exclusion criteria, 28 proteins remained. For each protein quantification, a surrogate peptide was chosen by first selecting a peptide pair with the best Pearson’s correlation coefficient, followed by empirically selecting the final peptide based on a good chromatographic peak. Quantification was performed using the sum of peak areas obtained for each transition of each surrogate peptide, and at least 4 transitions per peptide were used. The 28 surrogate peptides chosen for HDL proteins are highlighted in table 1.
Acetylation of LDL
LDL was acetylated as previously described by Basu et al. . Samples were extensively dialyzed before incubation with macrophages.
Measurement of 14C-cholesterol efflux
This study was approved by the Institutional Animal Care and Research Advisory Committee (#1015/2018) and was performed following the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6J mice were housed in a conventional animal facility at 22±2°C under a 12-h light/dark cycle with free access to commercial chow (Nuvilab CR1, São Paulo, Brazil) and drinking water. Bone marrow-derived cells were isolated from male, 6-week-old mice, and macrophages were differentiated . Briefly, tissues from the femur and tibias were cleaned and isolated at the knee joint. A needle size 26 and ½ and a 20-mL syringe filled with bone marrow medium (low-glucose DMEM with 0.8% penicillin/streptomycin, 10% heat-inactivated fetal calf serum, and 10% L929 cell-conditioned medium) were utilized to cut the end of each bone and to expel the bone marrow from both ends of the bones. Bone marrow was aspirated and expelled by utilizing a needle size 18 and ½ attached to a 20-mL syringe. Cells were centrifuged (6 min, 1,000 rpm at room temperature), resuspended in bone marrow medium, plated in culture dishes, and incubated for 5 days at 37°C under 5% (v/v) CO2. Then, the medium was changed to low-glucose DMEM containing 1% penicillin/streptomycin + 10% heat-inactivated fetal calf serum.
Bone marrow-derived macrophages (BMDMs) were overloaded with acetylated LDL (50 µg/mL DMEM) and 14C-cholesterol (0.3 µCi/mL) for 48 h. HDL particles from controls and subjects with DKD (50 µg/mL) were utilized as cholesterol acceptors in 6-h incubations, and the percentage of cholesterol efflux was calculated as 14C-cholesterol in the media/14C-cholesterol in the medium + 14C-cholesterol in cells x 100. Control incubations were performed in the presence of DMEM containing fatty-acid-free albumin (FAFA) in the absence of HDL, and the results were subtracted from those obtained in the presence of HDL, as previously described .
Measurement of HDL antioxidant activity
The ability of HDL from controls and individuals with DKD to inhibit LDL oxidation was determined by incubation of LDL (40 µg/mL) isolated from a unique healthy plasma donor with CuSO4 solution (1 mL; final concentration 10 µmol/L) in the presence of HDL (80 µg/mL). Lipoproteins were dialyzed against PBS without EDTA prior to incubation. The absorbance at 234 nm was continuously monitored every 3 min for 4 h, and the lag time phase for LDL oxidation (min) and the maximum ratio of conjugated diene formation were calculated .
Measurement of HDL anti-inflammatory activity
BMDMs were isolated and cultured as described above and then overloaded with acetylated LDL (50 µg/mL DMEM) and treated for 24 h with HDL (50 µg/mL DMEM) from controls and subjects with DKD. After washing, macrophages were incubated with lipopolysaccharide (LPS; 1 µg/mL DMEM) for 24 h. Medium was collected, and the amount of TNF-alpha and interleukin-6 (IL-6) was determined by ELISA (R&D System-Duo Set, Minneapolis, EUA) .
Statistical analysis was performed using the GraphPad Prism 5 program (GraphPad Software, Inc. 2007). Comparisons were made by the Kruskal-Wallis test with the Holm-Sidak posttest, Mann-Whitney or Student’s t test and Spearman’s linear correlation as appropriate. A value of P < 0.05 was considered statistically significant.