Renal ischemia/reperfusion (I/R) is a major problem leading to kidney damage following renal transplantation or major vascular surgery. Our laboratory has demonstrated that the major antioxidant in the mitochondria, manganese superoxide dismutase (MnSOD), is inactivated during renal transplantation (human and rodent) and renal I/R. These data suggested that the loss of MnSOD activity may be one key event that results in subsequent renal dysfunction, which is supported by preliminary data showing that induction of MnSOD (via gene delivery and estradiol pretreatment) protects the kidney from I/R injury. Conversely, compelling new data show that downregulation of MnSOD (using MnSOD heterozygote (-/+) transgenic mice) results in augmentation of mitochondrial and renal injury. Inactivation of MnSOD results in mitochondrial generation of superoxide and presumably mitochondrial damage;however, the mechanistic pathways involved with this injury remain unknown. Exciting new studies which focused on the five mitochondrial electron transport complexes, revealed alterations in Complexes III, IV, and V following renal I/R, which would also contribute to mitochondrial oxidant production. Thus, we hypothesize that: Electron transport complexes are targets of mitochondrial oxidant damage during I/R and that damage to specific complexes are the critical downstream event(s) that result from inactivation of MnSOD. We will use novel transgenic mouse models and renal cells designed to bi- directionally modulate MnSOD expression, along with cutting-edge proteomic analysis that will lead to identification of key mitochondrial targets that play a fundamental role in injury following renal I/R. Hypothesis 1. Even modest reductions in MnSOD activity (partial knockdown) lead to mitochondrial complex damage due to increased oxidant production following renal I/R. To test this hypothesis, MnSOD knockdown (using siRNA technology and mutant mice) will be combined with measurements of oxidant generation, mitochondrial integrity, cell viability, renal function, and mitochondrial proteomic analyses to determine the precise targets (complexes and/or subunits of complexes) and pathways involved with mitochondrial complex damage following MnSOD knockdown and I/R. Hypothesis 2. Increased MnSOD activity reduces oxidant production, restores normal mitochondrial complex function, and blunts renal injury following I/R. To test this hypothesis, MnSOD overexpression (using gene delivery, transgenic mice, and estradiol-mediated induction) will be combined with measurements of oxidant generation, cell viability, renal function, and mitochondrial proteomic analyses to determine the mechanisms that mediate protection from I/R injury due to MnSOD induction. Hypothesis 3. The new generation catalytic antioxidant manganese porphyrin (MnP) blunts renal injury and MnSOD inactivation during I/R via stabilization of mitochondrial electron transport complexes. Our recent published studies show that the long-term (24 hr) pretreatment of rats with MnP significantly improved MnSOD activity and renal function during I/R (Appendix 2). New studies will determine whether MnP prevents mitochondrial superoxide production during ischemia by preserving the integrity of the mitochondrial electron transport complexes, hence maintaining normal mitochondrial ATP levels.

Public Health Relevance

/RELEVANCE: The focus of this project is to determine how increased mitochondrial oxidants lead to renal injury after ischemia/reperfusion. Maintenance of adequate mitochondrial electron complex function is essential for normal ATP production. The proposed studies will, for the first time, identify modifications of key mitochondrial complex proteins during renal I/R, and vigorously characterize the MnSOD-dependent mechanisms that offer protection. Finally, the therapeutic potential of two reagents (estradiol and manganese porphyrin), which increase renal MnSOD activity, will be evaluated to set a basis for translational work relevant to renal transplantation. In summary, these findings may provide insight into other pathologic conditions involving mitochondrial oxidant production including atherosclerosis, stroke, neurodegenerative diseases, aging, and sepsis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK078936-04
Application #
8206848
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Hoshizaki, Deborah K
Project Start
2009-01-16
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
4
Fiscal Year
2012
Total Cost
$309,099
Indirect Cost
$95,927
Name
University of Arkansas for Medical Sciences
Department
Pharmacology
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
Shrum, S; MacMillan-Crow, L A; Parajuli, N (2016) Cold Storage Exacerbates Renal and Mitochondrial Dysfunction Following Transplantation. J Kidney 2:
Marine, Akira; Krager, Kimberly J; Aykin-Burns, Nukhet et al. (2014) Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells. Redox Biol 2:348-57
Marecki, John C; Parajuli, Nirmala; Crow, John P et al. (2014) The use of the Cre/loxP system to study oxidative stress in tissue-specific manganese superoxide dismutase knockout models. Antioxid Redox Signal 20:1655-70
Parajuli, Nirmala; MacMillan-Crow, Lee Ann (2013) Role of reduced manganese superoxide dismutase in ischemia-reperfusion injury: a possible trigger for autophagy and mitochondrial biogenesis? Am J Physiol Renal Physiol 304:F257-67
Patil, Naeem K; Saba, Hamida; MacMillan-Crow, Lee Ann (2013) Effect of S-nitrosoglutathione on renal mitochondrial function: a new mechanism for reversible regulation of manganese superoxide dismutase activity? Free Radic Biol Med 56:54-63
Mayeux, Philip R; MacMillan-Crow, Lee Ann (2012) Pharmacological targets in the renal peritubular microenvironment: implications for therapy for sepsis-induced acute kidney injury. Pharmacol Ther 134:139-55
Mitchell, Tanecia; Rotaru, Dumitru; Saba, Hamida et al. (2011) The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renal tubular cells and rat kidneys. J Pharmacol Exp Ther 336:682-92
MacMillan-Crow, Lee Ann; Crow, John P (2011) Does more MnSOD mean more hydrogen peroxide? Anticancer Agents Med Chem 11:178-80
Parajuli, Nirmala; Marine, Akira; Simmons, Sloane et al. (2011) Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse. Free Radic Biol Med 51:406-16
Mitchell, Tanecia; Saba, Hamida; Laakman, Joe et al. (2010) Role of mitochondrial-derived oxidants in renal tubular cell cold-storage injury. Free Radic Biol Med 49:1273-82