Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells

Abstract

Superoxide is widely regarded as the primary reactive oxygen species (ROS) which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK) cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ), a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS) inhibitor demonstrated that peroxynitrite (at low micromolar levels) induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation.

Keywords: Mitochondrial biogenesis; MnSOD; Peroxynitrite; Respiration; mtDNA; siRNA.

Graphical abstract

Fig. 1

Transient MnSOD knockdown in NRK cells. (A). MnSOD western blot after transfection (48 h) with 0–25 nM MnSOD siRNA. β-Actin was used as a loading control. (B) MnSOD western blot showing time course after transfection with 25 nM MnSOD siRNA. Graphs represent values after densitometric quantification of western blot results. (C) Representative MnSOD immunocytochemistry image showing decreased MnSOD expression after knockdown (KD) (25 nM siRNA; 48 h). Red stains for MnSOD, and blue DAPI stains for nuclei. (D) MnSOD activity decreased at 24 h following MnSOD KD, further decreased at 48 h and recovered to control level at 72 h. Control cells were treated with 25 nM nonsense siRNA. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells; #p<0.05 compared to 24hr treated cells.

Fig. 2

Superoxide and nitrotyrosine (NT) increase following MnSOD knockdown. (A) Representative images showing transient increase of MitoSOX Red (mitochondrial superoxide) fluorescence after siRNA transfection/knockdown (KD). (B) Representative nitrotyrosine immunocytochemistry image showing increased NT expression after MnSOD knockdown. DAPI stains nuclei blue. (C) Graphs showing quantification based on fluorescent intensity, arbitrary units. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells.

Fig. 3

Increased mitochondrial function following MnSOD knockdown. (A) ATP production increased significantly at 24 h, peaked at 48 h, and returned to control levels at 72 h post MnSOD knockdown. (*p<0.05 compared to control; #p<0.05 compared to 24 h treated cells; n=7). (B) Bioenergetics using Seahorse extracellular flux analyzer showing increased basal oxygen consumption rate (OCR) and reserve capacity following MnSOD KD (48 h post-transfection). (*p<0.05 compared to control, n=3) (C) Representative image showing increased MitoTracker Green fluorescence (indicating increased mitochondrial mass) after MnSOD KD (48 h). (D) Graph showing quantification based on fluorescent intensity, arbitrary units. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells.

Fig. 4

Markers of mitochondrial biogenesis increase following MnSOD knockdown. (A) Western blot analysis showing transiently increased PGC1α and Core II expression following MnSOD KD. β-Actin was used as a loading control. (B) mtDNA assessment using long range (LR) PCR as well as short fragment PCR (D-Loop and ND4). β-Actin was used as a nuclear encoded control in the PCR reactions. Graphs represent values after densitometric quantification of western blot or agarose gel results. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells; #p<0.05 compared to 24hr treated cells.

Fig. 5

MitoQ and L-NAME block oxidant generation following MnSOD knockdown. (A). MitoQ (0.1 μM), but not L-NAME (50 μM) prevented the mitochondrial superoxide (measured using MitoSOX Red fluorescence) increase following MnSOD knockdown (KD). (B) Both MitoQ (0.1 μM) and L-NAME (50 μM) prevented the increase in nitrotyrosine (NT) levels following MnSOD knockdown. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells.

Fig. 6

MitoQ and L-NAME block mitochondrial biogenesis following MnSOD knockdown. (A) Both MitoQ (0.1 μM) and L-NAME (50 μM) prevented the increase in OCR using Seahorse extracellular flux analysis following MnSOD knockdown (KD). No significant changes were observed in ECAR values. (B) Both MitoQ (0.1 μM) and L-NAME (50 μM) blocked the increase in LR mtDNA and mtDNA copy number (as described in Fig. 4) following MnSOD KD. (C) MitoQ (0.1 μM) treated cells prevented the increased protein expression of PGC1α or CORE II following MnSOD KD. L-NAME (50 μM) also blocked the increase of both PGC1α and CORE II at 48 h, but only CORE II at 24 h. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells.

Fig. 7

Dose dependent changes in mtDNA integrity and copy numbers following peroxynitrite treatment. (A) Representative PCR gel showing increases in long range (LR) mtDNA product (integrity) and short fragments (D-Loop and ND4; mtDNA copy number) following treatment with peroxynitrite (ONOO) (0–50 µM). Cells were treated with ONOO for 10 min, washed, and harvested 24 h later. β-Actin was used as a nuclear encoded control in the PCR reactions. (B) Graphs represent values after densitometric quantification of agarose gel results. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells.

Fig. 8

Peroxynitrite alters biogenesis markers and ATP levels. (A) Western blot analysis showing a dose dependent increase in PGC1α and Core II expression following peroxynitrite (ONOO; 0–50 µM) treatment. β-Actin was used as a loading control. Graphs represent values after densitometric quantification of western blot results. (B) Lower doses of peroxynitrite (ONOO; 0.5–1 µM) treatment lead to an increase in ATP production, while higher doses reduced levels. All data shown are mean±SEM (n=7). *p<0.05 compared to control cells.