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Abstract
Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.
Highlights
Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN)
Figure 2. 2-D proteome map of differentially expressed mitochondrial proteins. (A–C) Representative 2-D gels, stained with Deep Purple fluorescence dye, of mitochondrial proteins under normal glucose, high glucose, and osmotic control, respectively (n = 5 gels were derived from 5 individual culture flasks for each condition; a total of 15 gels were subjected to comparative analysis)
Mitochondria were isolated from Madin-Darby canine kidney (MDCK) renal tubular cells by sonication followed by differential centrifugation as detailed in “Materials and Methods”
Summary
Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). High glucose increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN. Recent studies have demonstrated that hyperglycemia induces mitochondrial fission and fragmentation in renal cells that are associated with ROS overproduction, increased mitophagy and apoptosis[8,9] These lines of evidence indicate that high glucose causes mitochondrial dysfunction leading to renal cell injury. Proteins were identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and subjected to analysis for potential post-translational modifications (PTMs) followed by validation with various assays
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