Original ContributionMitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells
Section snippets
Cell model and transfection studies
Normal rat kidney proximal tubular cells (NRK-52E; ATCC No. CRL-1571) were maintained in a humidified incubator gassed with 5% CO2, 95% air at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 5% fetal calf serum. Cells were grown to 60% confluency and divided into four treatment groups: (1) control, (2) high glucose, (3) MnSOD overexpression control, and (4) high glucose + MnSOD overexpression.
For MnSOD overexpression (Groups 3 and 4), NRK cells were transiently transfected with the
High glucose exposure does not alter MnSOD expression or activity in renal tubular cells
Exposure of NRK cells to high glucose (HG; 25 mM; 48 h) did not alter endogenous MnSOD protein or activity (Figs. 1A and B, pMnSOD). One goal of these studies was to determine the role that mitochondrial superoxide had in hyperglycemic damage; thus we engineered NRK cells that transiently overexpressed MnSOD. Our results showed an increased MnSOD expression (Fig. 1A) in NRK cells transfected with pMnSOD, which was further confirmed by an activity assay that indicated a 1.8-fold increase in
Acknowledgments
The authors thank Dr. Hamida Saba for her technical assistance with transfection studies and Ms. Tanecia Mitchell for her help with editing the manuscript. This research study was funded in part by a Pilot grant and the Graduate Student Research Fund from the University of Arkansas for Medical Sciences.
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