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. 2013 Feb 1;304(3):F257-67.
doi: 10.1152/ajprenal.00435.2012. Epub 2012 Nov 28.

Role of reduced manganese superoxide dismutase in ischemia-reperfusion injury: a possible trigger for autophagy and mitochondrial biogenesis?

Nirmala Parajuli  1 , Lee Ann MacMillan-Crow
Affiliations

Role of reduced manganese superoxide dismutase in ischemia-reperfusion injury: a possible trigger for autophagy and mitochondrial biogenesis?

Nirmala Parajuli et al. Am J Physiol Renal Physiol. .

Abstract

Excessive generation of superoxide and mitochondrial dysfunction has been described as being important events during ischemia-reperfusion (I/R) injury. Our laboratory has demonstrated that manganese superoxide dismutase (MnSOD), a major mitochondrial antioxidant that eliminates superoxide, is inactivated during renal transplantation and renal I/R and precedes development of renal failure. We hypothesized that MnSOD knockdown in the kidney augments renal damage during renal I/R. Using newly characterized kidney-specific MnSOD knockout (KO) mice the extent of renal damage and oxidant production after I/R was evaluated. These KO mice (without I/R) exhibited low expression and activity of MnSOD in the distal nephrons, had altered renal morphology, increased oxidant production, but surprisingly showed no alteration in renal function. After I/R the MnSOD KO mice showed similar levels of injury to the distal nephrons when compared with wild-type mice. Moreover, renal function, MnSOD activity, and tubular cell death were not significantly altered between the two genotypes after I/R. Interestingly, MnSOD KO alone increased autophagosome formation, mitochondrial biogenesis, and DNA replication/repair within the distal nephrons. These findings suggest that the chronic oxidative stress as a result of MnSOD knockdown induced multiple coordinated cell survival signals including autophagy and mitochondrial biogenesis, which protected the kidney against the acute oxidative stress following I/R.

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Figures

Fig. 1.
Fig. 1.
A: effect of manganese superoxide dismutase (MnSOD) knockdown on serum creatinine levels following I/R. Serum creatinine was determined in wild-type (WT) and MnSOD knockout (KO) mice after receiving 40 min ischemia and 18 h reperfusion. Values are expressed as means ± SE [n = 7 for sham (Sh); n = 7 for ischemia-reperfusion (I/R)]. B: neutrophil gelatinase-associated lipocalin (NGAL) expression following I/R injury. Ba: representative 200× micrographs show significant levels of NGAL expression in cortical and medullar regions of mouse kidney following mild (20 min) and severe (40 min) I/R. Bb: expression level of NGAL was evaluated semiquantitatively and scored. Error bar indicates mean ± SE (n = 4 for sham; n = 6 for I/R). *Means are significantly different, P < 0.05.
Fig. 2.
Fig. 2.
Effect of MnSOD knockdown on total tubular injury after I/R. A: representative 400× micrographs of periodic acid-Schiff (PAS) staining in renal cortex and medulla of WT and KO mice after sham and I/R surgery (asterisks, casts in lumen; arrowhead, tubular necrosis; thin arrow, cell swelling). Representative bar indicates 100 μm. Abbreviations: G, glomerulus; PT, proximal tubules; DN, distal nephrons. B: graph shows the pathological scoring for total tubular injury including casts in lumen, tubular dilation, loss of brush border, tubular necrosis, cell swelling, and epithelial cell flattening. Error bar indicates mean ± SE (n = 4 for sham; n = 6 for I/R). C: tubular injury score in proximal tubules and distal nephrons (distal tubule, loops of Henle, and collecting ducts). Error bar indicates mean ± SE (n = 4 for sham; n = 6 for I/R). *Means are significantly different, P < 0.05.
Fig. 3.
Fig. 3.
MnSOD activity following I/R. MnSOD activity was determined in total renal homogenates using the cytochrome c reduction method. WT mice showed significant MnSOD activity loss after I/R. MnSOD KO mice, which showed ∼60% activity loss prior to I/R, did not decline further after I/R. Error bar represents mean ± SE (n = 3 for sham; n = 6 for I/R). *Means are significantly different, P < 0.05.
Fig. 4.
Fig. 4.
Effect of MnSOD knockdown on protein nitration (oxidative stress) after I/R. A, a–o: representative 400× micrographs of nitrotyrosine immunostaining are shown. The specificity of nitrotyrosine binding in the renal tissue was confirmed by blocking the antibody with 3′-nitrotyrosine (10 mM) using sections of KO kidney after I/R (m–o). Bar indicates 100 μm. B: expression level of nitrotyrosine was evaluated semiquantitatively and scored. Error bar indicates mean ± SE (n = 4 for sham; n = 6 for I/R). *Means are significantly different, P < 0.05.
Fig. 5.
Fig. 5.
MnSOD knockdown failed to augment apoptosis induction after I/R. A, a–l: representative 400× micrographs show TUNEL-positive brown nuclei (apoptotic cells) from cortex, outer medulla and inner medulla regions of sham and I/R kidneys. Bar indicates 100 μm. B: TUNEL-positive cells were counted in 7 different fields (200×), and the average was reported (left panel). Error bar indicates mean ± SE (n = 3 for sham; n = 5 for I/R). Right panel shows tabulation of TUNEL-positive nuclei counted in different segments of nephrons, namely proximal tubules (proximal tubules and glomerulus) and distal nephrons (distal tubules, loops of Henle, and collecting ducts). *Means are significantly different, P < 0.05.
Fig. 6.
Fig. 6.
MnSOD knockdown triggers autophagosome formation in sham kidneys. A: a–f show representative micrographs of total LC 3 (I and II) immunostaining in WT and MnSOD KO sham kidneys; g–l shows representative micrographs of MnSOD expression in sham kidneys. Bar indicates 100 μm. B: Western blot detection for LC3 I and II from renal homogenate of control kidneys. GAPDH was used as a loading control. Graph represents values after densitometric quantification (LC3II/LC3I) of Western blot results. LC3 II is increased in MnSOD KO sham kidneys. C: Western blot detection for Atg5 from renal homogenate of control kidneys. GAPDH was used as a loading control. Atg5 is increased in MnSOD KO sham kidneys. Graph represents values after densitometric quantification of Western blot results. Error bar indicates mean ± SE (n = 3). *Means are significantly different, P < 0.05.
Fig. 7.
Fig. 7.
Mitochondrial biogenesis following MnSOD knockdown. A: Representative micrographs of mitochondrial proteins (n = 6); a–f show Complex IV subunit I (COXI, mitochondrial DNA encoded), and g–l show Complex V subunit (ATP5B, nuclear DNA encoded). Representative bar indicates 100 μm. B: citrate synthase activity showed ∼20% increase in MnSOD KO control kidney compared with the WT control kidney. Error bar indicates mean ± SE (n = 5). *Means are significantly different, P < 0.05.
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