Antibodies used in these studies were as follows: human-specific active fragment of caspase 9 and human cytochrome-c (Cell Signaling, Beverley MA,), human active fragment caspase-8 and caspase-2 (BD Biosciences), endonuclease G (Chemicon, Temecula, CA), and β-Actin (Sigma, #A5316). Cell-permeable inhibitors of caspase 9 (Z-LEHD-FMK) and caspase-8 (Z-IETD-FMK) were from R&D systems (Minneapolis, MN). Etomoxir and 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) were from Sigma and Toronto Research Chemicals (Ontario, Canada), respectively.
Preparation of FFA-albumin complexes
Fatty acid-albumin solutions were prepared by the protocol of Spector . Briefly, sodium salts of FFA (Nu-Chek Prep, Elysian, MN) were added to PBS and gently warmed to facilitate solubility without damaging the fatty acid . The warm, clear fatty acid salt solution was complexed to 5% fatty acid-free BSA in PBS at a 6:1 fatty acid to BSA molar ratio. The sterile filtered, complexed fatty acid solution was added to the serum-containing cell culture medium to obtain the indicated final FFA concentration. The final FFA concentration in the medium was confirmed with an enzymatic colorimetric assay (NEFA C, Wako). We also confirmed that addition of the complex to culture medium did not significantly alter the pH.
Apoptotic cell death in cultured human mesangial cells
Human mesangial cells (HMC), purchased from Cambrex Bioscience Inc. (Walkersville, MD), were maintained in Dulbecco's modified essential medium (Gibco-BRL) supplemented with 17% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 5 ng/ml selenite, and 5 μg/ml each of insulin and transferrin. Characterization was performed by phase contrast microscopy and by immunostaining for intermediate filaments and surface antigens as described previously . Briefly, cells were positive for desmin, vimentin, and myosin, but did not stain for factor VIII, keratin, or common leukocyte antigen.
To measure endogenous levels of cleaved caspase-3, cells were lysed in a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml luepeptin. After adjusting for cell protein (DC Assay, BioRad, Hercules, CA), the amount of cleaved human caspase-3 (Asp 175) was measured by ELISA (Cell Signaling). For Western blotting of cleaved caspase proteins, the cells were washed with ice cold PBS and scraped in CHAPS extraction buffer(50 mM Pipes/HCI, pH 6.5, 2 mM EDTA, 0.1% Chaps, 20 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 5 mM DTT, 2 mM Na pyrophosphate, 1 mM Na3VO4, and 1 mM NaF) and centrifuged at 2,000 × g for 10 min at 4°C. Protein content in the supernatant was assayed with the DC protein assay. An aliquot of the lysate (25 μg protein) was boiled in SDS sample buffer, resolved on a 4–12% SDS-PAGE gradient gel, and transferred to a 0.2 μm nitrocellulose membrane. After blocking in 5% non-fat dried milk in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h, the membrane was washed 3 times with TBS-T for 5 min each and incubated overnight at 4°C with primary antibody in 3% BSA in TBS-T. After incubating with suitable HRP-labeled secondary Ab (1:2,000) and extensive washing, the proteins were detected by chemiluminescence with an average exposure ranging from 10–30 sec. As previously described , the Western blots were analyzed by densitometry in NIH Image by normalizing values for the relevant caspase fragment to the highest value within each experiments (maximum level = 1).
To quantify the number of pyknotic nuclei, HMC on coverslips were washed once with PBS and fixed for 20 min with freshly-prepared 3.7% formaldehyde/20% sucrose in PBS. After washing twice with PBS the HMC were stained with 5 μg/ml Hoechst 33342 (Molecular Probes, Eugene OR) and mounted in Slow Fade Light (Molecular Probes). Using a Nikon Diaphot microscope, the number of pyknotic nuclei were counted and expressed as a percentage of the total number of nuclei counted (n = >300 nuclei per condition).
DNA fragmentation was assessed by measuring release of nucleosomal fragments into the cytosol (Cell Death Detection ELISA Plus, Roche). Briefly, HMC in 24-well plates were centrifuged in situ for 10 min at 200 × g and the supernatant gently removed. The monolayer was incubated in lysis buffer for 30 min at room temperature and centrifuged again at 200 × g. The supernatant (i.e., cytosolic fraction) was assayed immediately for nucleosomal fragments.
Enzymatic assay of caspase 9 in HMC
HMC treated with FFA were lysed (20 mM Tris, pH 7.5, 150 mM NaCl, 1.0% Triton X-100) and frozen at -40°C. Equivalent amounts of total HMC protein were added to buffer containing the LEHD caspase 9 peptide substrate linked to a cleavable luciferase substrate, aminoluciferin (Promega). The amount of light produced in a coupled reaction with luciferase was measured once every hour for 3 hours in a Berthold Luminometer. Experiments with increasing amounts of cell protein confirmed that the assay was in the linear range under the conditions described.
Measurements of cytochrome -c and endonuclease G redistribution
To analyze cytochrome-c redistribution in HMC treated with FFA, cells were fractionated into cytosol and membrane fractions using 0.05% digitonin in an isotonic sucrose buffer exactly as described by Dong and coworkers . Because cytochrome-c release occurs mostly from mitochondria, Western blot analysis of cytosol and membrane fractions is expected to reflect cytochrome-c translocation from mitochondria to the cytoplasm. In separate experiments, the same protocol was used to assess release into the cytoplasm of endonuclease G.