These residues that can react with HNE may not affect the binding of apo A-I with lipids but with other proteins involved in HDL metabolism (see Figure 3B, residues K47, K83, H216, K250 and K262); in contrast, some histidine and lysine residues (residues K64, K130, H179, H186, and H223) interact tightly with lipids in the internal side of the complex. it overnight at 37C with 100 for 10 minutes to isolate plasma and were Ferroquine then stored at ?80C until use (Supplemental Methods). Animal Procedures All experiments were performed under the authorization number 69-266-0501 and agreed with the guidelines laid down by the French Ministry of Agriculture (number 2013-118) and the European Union Council Directive for the protection of animals used for scientific purposes of September 22, 2010 (2010/63UE). Adult male white New Zealand rabbits (CEGAVssc, Saint Mars dEgrenne, France) were housed in individual cages at constant ambient heat (21C23C) and humidity (45%C50%) with a 12-hour light cycle. All animals had free access to tap water. After a 7-day period of acclimation, rabbits were randomized to either the 5/6 nephrectomy group or the control group. Nephrectomy was performed as described by Gotloib for 2 minutes and stored at ?20C. The serum iohexol concentration was measured by HPLC as previously described38 and GFR was calculated using a bicompartmental model equation. Euthanasia, Necropsy, and Renal Histology At the end of the GFR measurement, rabbits were deeply anesthetized with an overdose of sodium pentobarbital (70 mg/kg, intravenously). Blood was removed by cardiac puncture and placed in EDTA-coated tubes. After a centrifugation at 1250 for 10 minutes, plasma was aliquoted and stored at ?80C. Urine was obtained a direct bladder puncture and stored at ?20C. Kidneys were removed, weighed, and stored in buffered formalin 10% (w/v) for histologic examination. Kidneys were dehydrated using ethanol, embedded in paraffin, and sliced. Hematoxylin Erythrosine Saffron and Sirius Red stainings were performed. Isolation of Lipoproteins from the Plasma Lipoproteins were separated from plasma by stepwise potassium bromide (KBr) density gradient ultracentrifugation as described by Ferroquine Havel for 3 hours 30 minutes at 15C was performed to remove the top layer corresponding to VLDL and chylomicrons. Then, the plasma density was adjusted to 1 1.063 g/ml with KBr (M=119.01 g/mol). After a second centrifugation at 100,000 for 5 hours at 4C, the GADD45gamma orange Ferroquine ring, corresponding to LDL, was collected. Finally, plasma density was adjusted to 1 1.21 g/ml with KBr and, after centrifugation at 100,000 for 6 hours 30 minutes at 4C, the orange ring corresponding to HDL was collected. For the isolation of all of the lipoproteins together, a single ultracentrifugation was performed at 100,000 for 6 hours 30 minutes at 4C after an adjustment of plasma density to 1 1.21 g/ml with KBr. For platelet aggregation and the copper-induced HDL oxidation assay, freshly isolated HDL samples were used to prevent HDL ultrastructure modification due to freezing.40 After ultracentrifugation, lipoproteins were extensively dialyzed against PBS with 1 mM EDTA for 3 hours, twice at room temperature and then overnight at 4C. A last dialysis without EDTA was performed just before the platelet aggregation and copper-induced oxidation. HDL samples were stored at 4C for a maximum of 48 hours before the experiments. Biochemistry Serum creatinine measurement was performed using the Siemens enzymatic method (around the Dimension Vista System; Siemens Healthcare, Erlangen, Germany). Urea was measured using the urease test (Vista 1500). Cholesterol and triacylglycerol levels were measured using enzymatic kits (Biomerieux, Marcy lEtoile, France). HDL concentration was measured with an enzymatic kit (Abcam, Paris, France). Malondialdehyde (MDA) was measured by HPLC coupled to ultraviolet-visible detection (Diode Array Detector) as described by Grotto the PRIDE partner repository with the data set identifier PXD013301 (DOI, 10.6019/PXD013301; username, ku.ca.ibe@65462reweiver; password, oMD4gyDT). Molecular Modeling To study the accessibility of residues, the structure of truncated human apo A-I (Protein Data Lender [PDB] code 1av1) was downloaded from the PDB database.49 All observations were performed using PyMOL software by representing the test. Multiple comparisons were made with KruskalCWallis test and, whenever appropriate, Dunn assessments. Differences were considered as significant at Value(%)3 (21)17 (74)19 (95)?Stroke, (%)0 (0)2 (9)1 (5)?CHD, (%)0 (0)5 (22)3 (15)?Cardiopathy,.