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Two-dimensional fluorescence difference gel electrophoresis analysis of the urine proteome in human diabetic nephropathy

Authors

  • Kumar Sharma,

    Corresponding author
    1. Center for Diabetic Kidney Disease, Thomas Jefferson University, Philadelphia, PA, USA
    2. Dorrance Hamilton Research Laboratories, Thomas Jefferson University, Philadelphia, PA, USA
    • Thomas Jefferson University, Suite 353, Jeff Alumni Hall, 1020 Locust Street, Philadelphia, PA 19107, USA Fax: +1-215-923-7212
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  • SoHee Lee,

    1. Center for Diabetic Kidney Disease, Thomas Jefferson University, Philadelphia, PA, USA
    2. Dorrance Hamilton Research Laboratories, Thomas Jefferson University, Philadelphia, PA, USA
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  • Steven Han,

    1. Center for Diabetic Kidney Disease, Thomas Jefferson University, Philadelphia, PA, USA
    2. Dorrance Hamilton Research Laboratories, Thomas Jefferson University, Philadelphia, PA, USA
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  • Sungchun Lee,

    1. Center for Diabetic Kidney Disease, Thomas Jefferson University, Philadelphia, PA, USA
    2. Dorrance Hamilton Research Laboratories, Thomas Jefferson University, Philadelphia, PA, USA
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  • Barbara Francos,

    1. Center for Diabetic Kidney Disease, Thomas Jefferson University, Philadelphia, PA, USA
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  • Peter McCue,

    1. Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
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  • Richard Wassell,

    1. Proteomics Core Facility, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
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  • M. Alexander Shaw,

    1. Proteomics Core Facility, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
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  • Satish P. RamachandraRao

    1. Dorrance Hamilton Research Laboratories, Thomas Jefferson University, Philadelphia, PA, USA
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Abstract

Urinary proteins may provide clues regarding pathogenesis of kidney disease as well as providing markers of disease activity. We employed two-dimensional differential in-gel electrophoretic analysis (2-D DIGE) to assess multiple urine samples in patients with diabetic nephropathy. Patient samples were collected as timed overnight collections. All the patients had longstanding diabetes, impaired renal function, and overt proteinuria. Control and patient urinary protein were analyzed by 2-D DIGE and DeCyder analysis. Ninety-nine spots were significantly regulated in the urine proteome of the diabetic samples, with 63 up- and 36 down-regulated. One spot corresponding to a pI 5–6 and a molecular weight between 45 and 66 kDa was consistently up-regulated by 19-fold across individuals in the diabetic group. Surface-enhanced laser desorption/ionization-time of flight analysis of in-gel tryptic digest of this spot identified this protein as alpha 1 antitrypsin (AAT). ELISA of urine samples from a separate group of patients and controls confirmed a marked increase of AAT in diabetic patients. Immunostaining of human diabetic kidneys revealed up-regulation of AAT in areas of renal fibrosis. In conclusion, we developed a method to analyze numerous urine samples from patients and allowed for detection and identification of regulated urine protein spots.

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