Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.

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.

Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant Mn(III) meso-tetrakis(N-hexylpyridinium-2-yl)porphyrin, (MnTnHex-2-PyP5+) using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP5+ in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 hr protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 μg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified ATP synthase- beta subunit as a key protein induced by MnTnHex-2-PyP5+ treatment alone, and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP5+ protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.