Original Contribution
Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

https://doi.org/10.1016/j.freeradbiomed.2009.01.022Get rights and content

Abstract

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.

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.

References (47)

  • MacMillan-CrowL.A. et al.

    Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite

    Arch. Biochem. Biophys.

    (1999)
  • KorshunovS.S. et al.

    High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

    FEBS Lett.

    (1997)
  • RoloA.P. et al.

    Diabetes and mitochondrial function: role of hyperglycemia and oxidative stress

    Toxicol. Appl. Pharmacol.

    (2006)
  • NishikawaT. et al.

    The missing link: a single unifying mechanism for diabetic complications

    Kidney Int. Suppl.

    (2000)
  • TurrensJ.F. et al.

    Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria

    Arch. Biochem. Biophys.

    (1985)
  • ParadiesG. et al.

    Reactive oxygen species generated by the mitochondrial respiratory chain affect the complex III activity via cardiolipin peroxidation in beef-heart submitochondrial particles

    Mitochondrion

    (2001)
  • U.S. Renal Data System. 2008 Annual Report, in Atlas of End-Stage Renal Disease in the United States, Bethesda, MD,...
  • NishikawaT. et al.

    Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage

    Nature

    (2000)
  • DuX.L. et al.

    Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation

    Proc. Natl. Acad. Sci. USA

    (2000)
  • ForbesJ.M. et al.

    Oxidative stress as a major culprit in kidney disease in diabetes

    Diabetes

    (2008)
  • BrownleeM.

    Biochemistry and molecular cell biology of diabetic complications

    Nature

    (2001)
  • ShenX. et al.

    Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy

    Diabetes

    (2006)
  • BerteraS. et al.

    Gene transfer of manganese superoxide dismutase extends islet graft function in a mouse model of autoimmune diabetes

    Diabetes

    (2003)
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