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Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

Munusamy S, Saba H, Mitchell T, Megyesi JK, Brock RW, Macmillan-Crow LA - BMC Endocr Disord (2009)

Bottom Line: Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury.Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria.The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology & Toxicology, Division of Nephrology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. munusamyshankar@uams.edu

ABSTRACT

Background: Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).

Methods: Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.

Results: Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.

Conclusion: In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.

No MeSH data available.


Related in: MedlinePlus

Identification of renal Complex-III following BN-PAGE. Western blot analysis for Rieske Protein (A and C) and Core 2 Protein (B and D) to confirm the identity of the Complex-III band after one dimension BN-PAGE (left panel/A and B) and 2D-BN-PAGE (right panel/C and D) using control (C) and diabetic (D) rat kidney mitochondria.
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Figure 4: Identification of renal Complex-III following BN-PAGE. Western blot analysis for Rieske Protein (A and C) and Core 2 Protein (B and D) to confirm the identity of the Complex-III band after one dimension BN-PAGE (left panel/A and B) and 2D-BN-PAGE (right panel/C and D) using control (C) and diabetic (D) rat kidney mitochondria.

Mentions: Additional experiments were carried out to verify the identity of Complex III following BN-PAGE. These included Western blotting for Rieske and Core 2 proteins after BN-PAGE (Figure 4A &4B) as well as BN-PAGE followed by SDS-PAGE in the second dimension (2D BN-PAGE) (Figure 4C &4D). These data clearly demonstrated that the band identified as Complex III in the BN-PAGE contains both the Rieske and Core 2 subunits of Complex III.


Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

Munusamy S, Saba H, Mitchell T, Megyesi JK, Brock RW, Macmillan-Crow LA - BMC Endocr Disord (2009)

Identification of renal Complex-III following BN-PAGE. Western blot analysis for Rieske Protein (A and C) and Core 2 Protein (B and D) to confirm the identity of the Complex-III band after one dimension BN-PAGE (left panel/A and B) and 2D-BN-PAGE (right panel/C and D) using control (C) and diabetic (D) rat kidney mitochondria.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2636815&req=5

Figure 4: Identification of renal Complex-III following BN-PAGE. Western blot analysis for Rieske Protein (A and C) and Core 2 Protein (B and D) to confirm the identity of the Complex-III band after one dimension BN-PAGE (left panel/A and B) and 2D-BN-PAGE (right panel/C and D) using control (C) and diabetic (D) rat kidney mitochondria.
Mentions: Additional experiments were carried out to verify the identity of Complex III following BN-PAGE. These included Western blotting for Rieske and Core 2 proteins after BN-PAGE (Figure 4A &4B) as well as BN-PAGE followed by SDS-PAGE in the second dimension (2D BN-PAGE) (Figure 4C &4D). These data clearly demonstrated that the band identified as Complex III in the BN-PAGE contains both the Rieske and Core 2 subunits of Complex III.

Bottom Line: Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury.Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria.The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology & Toxicology, Division of Nephrology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. munusamyshankar@uams.edu

ABSTRACT

Background: Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection).

Methods: Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model.

Results: Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes.

Conclusion: In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.

No MeSH data available.


Related in: MedlinePlus