Diabetes-Induced Changes in the Renal Cortical Proteome Assessed with 2D Gel Electrophoresis and Mass Spectrometry.

To understand the spectrum of proteins affected by type 2 diabetes and to characterize molecular functions and biological processes they control, we analyzed the renal cortical proteome of 3 month hyperglycemic db/db mice and age-matched congenic controls. Predominantly hydrophilic proteins were extracted from the cytosolic compartment of buffer (containing protease and phosphatase inhibitors)-perfused renal cortex, then equal amounts of extract pooled from 5 control and 5 diabetic mice were subjected to 2D gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF, and LC-MS/MS. The staining intensity of approximately one third of all protein spots on 2D gels was affected by diabetes. This approach yielded 278 high confidence identifications whose expression levels were significantly increased or decreased >2 fold by diabetes, of which 170 mapped to gene identifiers representing 147 non-redundant proteins. Based on Gene Ontology classification, 80% of these proteins modulated physiological functions, 55% involved metabolism, ∼25% involved carboxylic and organic acid metabolism, 14% involved biosynthesis or catabolism, and 12% involved fatty acid metabolism. Most of these proteins (∼85%) were increased in abundance. Predominant molecular functions were catalytic (61%), oxidoreductase (20%), and transferase (17%) activities, and nucleotide and ATP binding (11 - 15%). Mitochondrial proteins accounted for 28% of the proteins identified as significantly altered by diabetes. The top-ranked network described by Ingenuity Pathway Analysis indicated PPARα was the most common node of interaction for the numerous enzymes whose expression levels were influenced by diabetes. These differentially regulated proteins create a foundation for a systems biology exploration of molecular mechanisms underlying the pathophysiology of diabetic nephropathy. ADA-Funded Research [ABSTRACT FROM AUTHOR]