These results indicate that renal Complex-III appears to be an early and specific mitochondrial target during experimental type-1 diabetes. Complex-III is centrally located in the electron transfer process and has been implicated as one of the major sites for superoxide generation in the mitochondria during diabetes [8
]. Partial inhibition of Complex-III during conditions of increased respiration [9
] would decrease the transfer of electrons from ubiquinol to Complex-III, and increase the half-life of ubisemiquinone, which leads to generation of superoxide [8
Intriguingly, our results indicated an upregulation of renal Complex-III subunits, Core 2 and Rieske protein during diabetes. However, using BN-PAGE it was shown that Complex III was not correctly assembled (decreased levels on BN-PAGE) which also correlated with a decrease in Complex-III activity. Thus, the paradoxical increase in expression of individual subunits, Core 2 and Rieske proteins could be a compensatory response to restore the activity of Complex-III during diabetes.
Rieske subunit is a nuclear encoded protein that is imported into the mitochondrial matrix where it undergoes a two-step cleavage of its pre-sequence and is translocated into the inner mitochondrial membrane. It has an iron-sulfur [2Fe-2S] redox center that centrally participates in the Q cycle by transferring an electron from ubiquinol to cytochrome c1
, which then donates it to cytochrome c. Core 2 subunit, which is also a nuclear encoded protein is a homolog of mitochondrial processing protease-alpha subunit and is thought to be important for mitochondrial protein import, processing and integrity of Complex-III [20
]. Although, Core 2 subunit is not primarily involved in electron transfer, genetic deletion of Core 2 has been shown to affect the assembly of Complex-III [21
]. Thus, an increase in the Rieske as well as the Core 2 subunits suggests a possible assembly defect in Complex-III.
To our knowledge, this is the first report demonstrating that renal Complex-III and its subunits are altered in the early stages of diabetes. A study by Rosca et al. showed previously that Complex-III was a target for glycation by methyl glyoxal and inactivated during chronic diabetes (12 months) [8
], while no significant changes were reported at 2 months of diabetes. Although, the reason for this discrepancy remains unknown; one difference could be the fraction of the kidney analyzed in the two studies. Our study utilized whole rat kidneys, whereas the study by Rosca et al. used the cortical fractions of kidneys. Although the renal cortex has been primarily considered to be the energetically active segment of the nephron, several studies suggest that the medullary thick ascending limb (mTAL) is an active site for reabsorption in kidney and thus might play a major role in mitochondrial superoxide production. In addition, increased oxidant production has been observed in both the cortical and medullar regions of the kidney. Thus, we felt it was important to include both cortex and medulla (whole kidney) for mitochondrial isolation and study the net effect of diabetes on the renal mitochondria.
Despite the findings by Katyare and Satav [9
] who showed increased cytochrome aa3 contents in renal mitochondria during diabetes, we did not detect any significant changes in Complex IV activity in our rat model of diabetes. It is also important to note that the authors could not correlate the changes in cytochrome content with the altered respiration rates observed during diabetes. In addition, these authors did not measure Complex IV activity and we did not measure cytochrome aa3 contents so a direct comparison of these studies is difficult to assess. Again, one important difference between the two studies is the source of renal mitochondria used: we used whole kidney mitochondria, while Katyare and Satav used cortical mitochondria as discussed above.
Our results also showed that F0
-ATPase activity and renal ATP levels were significantly increased in the diabetic renal mitochondria compared to controls. Interestingly, the study by Katyare and Satav [9
] also demonstrated that respiration and ATPase activity were increased in diabetic renal mitochondria. Thus, it is possible that the increased respiration leads to increased proton motive force (hyperpolarization) and increased ATP levels which serve to fulfill the increased energy demands of the kidney during diabetes. Concomitantly, the increased respiration would likely result in more superoxide (oxidant) generation which could lead to alterations in Complex III. In fact, we have observed that renal proximal tubule cells exposed to hyperglycemia (25 mM; 48 hr) results in mitochondrial hyperpolarization, Complex-III inhibition, and oxidant production (manuscript under review).