Correlation Between Autophagy-related Proteins and Inflammatory Markers in Hemodialysis Patients
Condition: Autophagy
Study type: Observational

Summary: 
      One recent study demonstrated impaired autophagy in patients receiving hemodialysis (HD). To
      clarify whether this alteration is related to the inflammatory state in HD patients, we
      focused on basal autophagy in peripheral blood mononuclear cells (PBMCs) of HD patients with
      and without inflammation and controls. PBMCs were harvested using Ficoll density gradient
      centrifugation . Levels of the autophagy-associated proteins ubiquitin-binding protein p62
      (p62), microtubule-associated proteins 1A/1B light chain 3A (LC3I/II) and beclin-1 in PBMCs
      will be detected by western blotting. Enzyme-linked immunosorbent assay kits will be used to
      detect the serum concentrations of interleukin (IL)-6, liposaccharide-binding protein (LBP)
      and tumor necrosis factor (TNF)-α.
    

Detailed description: 
      Study Design and Population

      Study Design We shall enroll 30 healthy volunteers as the control group. 180 sex- and
      age-matched hemodialysis patients will be enrolled in this study. To be included in the
      study, patients have to be at least 20-years-old and on outpatient hemodialysis (HD) for at
      least 3 months. Patients are excluded if they had malignancy, severe liver disease (bilirubin
      >1.6 mg/dl), uncontrolled hypertension, severe obesity (BMI >35), currently on carbamazepine,
      statins or immunosuppressive agents. The medical record is thoroughly reviewed for each
      subject by a collaborating physician in the study. All subjects will provide written informed
      consent to participate and the protocol will be send to the institutional review boards of
      Tungs' Taichung Metroharbour Hospital

      Study Parameters Predialysis blood samples are obtained on a mid-week day. Within 30 min
      after sampling, the remaining blood is centrifuged at 3,000 g for 10 min, immediately
      aliquoted and frozen at -80°C until further analysis.

      Cytokine assays The following markers of inflammation and hemostasis are determined by ELISA
      in patients' sera: interleukin-6 (Quantikine, R&D Systems,) and plasma concentrations of CRP
      in a highly sensitive assay (hsCRP).

      Analysis of lipids/lipoproteins Total cholesterol, highdensity lipoprotein (HDL) cholesterol,
      and triglyceride concentrations are determined enzymatically. Plasma glucose is measured by a
      glucose-oxidase method.

      Measurements of Liposaccharide-binding protein (LBP) The LBP was determined from serum
      samples and controls using standardized enzymelinked immunosorbent assay (ELISA) methods, and
      serum from normal control subjects was used for interassay variation.

      Isolation of PBMCs Ethylenediamine tetraacetic acid Vacutainer™ tubes are used to collect
      venous blood samples (10 ml) from fasting participants in the early morning. Aliquots of the
      supernatant are collected by centrifugation at 800 x g and 25˚C for 15 min and subsequently
      store at -80˚C. The remaining blood is mixed and add slowly dropwise to a centrifuge tube
      containing 10 ml of Ficoll separation medium. PBMCs are isolated according to manufacturer's
      protocol and store at -80˚C.

      Western blotting PBMCs are lysed with RIPA lysis buffer and protein concentrations determined
      with the BCA Protein Assay kit according to the manufacturer's protocol. Subsequently, 20 μg
      of protein/well are separated using 12% SDS-PAGE. The proteins are subsequently transferred
      to nitrocellulose membranes at 300 mA for 60 min. The nitrocellulose membranes are blocked
      with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% non-fat milk powder for 2 h
      at room temperature. The nitrocellulose membranes are incubated with primary antibodies
      overnight at 4˚C on a shaker set at a slow speed. The nitrocellulose membranes are washed
      thrice with TBST and incubated with secondary antibodies for 1 h at room temperature. After
      washing thrice, ECL substrate is added to the nitrocellulose membrane. The signal is detected
      using Image Lab™ software and band density will be quantified with imager software .

      The primary antibodies against microtubule-associated proteins 1A/1B light chain 3A ,
      ubiquitin-binding protein p62 and beclin-1 as well as GAPDH will be purchased.
      Peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies are used.
    

Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        patients have to be at least 20-years-old and on outpatient hemodialysis (HD) for at least
        3 months.
      
Keywords: autophagy; inflammatory markers