The present study documents the effects of SOD1 over-expression in an in vitro model of ATP depletion injury using novel lentiviral vector techniques to modify SOD1. To our knowledge, this is the first report using lentiviral vector mediated over-expression of SOD1 in renal epithelial cells to examine the effects of ATP depletion-recovery. The main findings of this study were the following: (1) SOD1 over-expression partially prevented cytotoxicity in LLC-PK1 cells following ATP depletion-recovery; (2) there was decreased nuclear fragmentation in SOD1 cells compared to EGFP cells following ATP depletion-recovery; (3) SOD1 over-expression partially attenuated caspase-3 activation; (4) the addition of catalase attenuated cytotoxicity and caspase-3 further, although catalase did not have significant synergistic protective effects with SOD1 and (5) the mitochondrially targeted antioxidant, MitoTEMPO was also effective in reducing cytotoxicity and caspase-3 activation in ATP depleted renal epithelial cells.
We achieved greater than 99% transduction efficiency by using the VSV-G pseudotyped replication-defective lentiviral vectors thus demonstrating this extremely efficient method of transducing LLC-PK1 cells. Further, we have shown an increase in SOD1 mRNA and protein using the lentiviral mediated transduction technique. There was also a corresponding increase in SOD1 activity in SOD1 over-expressing cells compared to EGFP although the levels were not as high as shown by the Western blot. This discrepancy in the protein versus activity levels could possibly be due to some posttranslational modification of the SOD1 protein in the transduced cells. Lentiviral vector are distinctly advantageous over other vectors currently in use, e.g., adenoviral vectors. Unlike adenoviral vectors which remain largely episomal, lentiviral vectors are integrated into the host genome. This ability allows for long term, stable transgene expression by lentiviral transduction compared to other viral vectors where there is a rapid loss of transgene expression. To our knowledge, this is the first report demonstrating the use of lentiviral vector mediated over-expression of SOD1 in LLC-PK1 cells.
Cytotoxicity evoked by the massive formation of radicals, NO•
, can be ascribed to apoptosis or necrosis, two defined features of the cell death program [36
]. Necrosis is regarded as accidental cell death [37
] with characteristic signs of cell swelling, membrane rupture, randomly digested DNA and cell dissolution [38
]. On the other hand, apoptosis is a regulated process with cells shrinking, chromatin condensation and DNA fragmentation. Whether cells undergo apoptosis or necrosis is dependant on ATP content [39
Our finding in the current study that 2–2 h ATP depletion-recovery did not increase cytotoxicity significantly over serum free controls is in agreement with previously published reports from our laboratory [22
]. Following longer duration of (4 h) of ATP depletion in vitro however, there was an increase in cytotoxicity that was partially attenuated by SOD1. SOD1 is the major O2
-disproportionating enzyme in the proximal tubular cells and thus it is likely that it contributes partially to the cellular protective effects following ATP depletion-recovery. However, SOD1 was not able to decrease LDH levels down to levels observed in serum free controls indicating that other radicals and mitochondrial sources of O2
are also involved in injury. In this regard, it has been previously shown that over-expression of the mitochondrial SOD2 (Mn SOD) prevents cell death [42
] and protects against IR injury in heart, liver or brain tissues [16
]. The second caveat with this model of injury is that it does not truly represent in vivo ischemia-reperfusion (IR) injury. It is important to note that other investigators have successfully used hypoxia-reoxygenation of proximal tubular cells as a surrogate model of IR [46
]. Future studies in our laboratory will also examine the role of SOD1 over-expression in a model of hypoxia-reoxygenation injury.
We also measured O2
•− levels by DHE fluorescence to determine whether SOD1 over-expression was sufficient to decrease ATP depletion-mediated increases in O2
•−. Although SOD1 over-expression decreased O2
•− following earlier time points of injury, the O2
•− levels in injured LLC-SOD1 cells were higher than the serum free controls. Following 4–2 h ATP depletion-recovery, SOD1 over-expression did not impact O2
•− levels. This result was not surprising to us because it is possible that at the longer time point of injury, O2
•− is increased to a maximal level, which SOD1 is unable to scavenge. Additionally it may be due to the likely activation of inner mitochondrial sources of O2
•− in our model of injury against which SOD1 is ineffective.
It has also been previously shown that ATP depletion increases potassium influx due to increase in activity of mitochondrial potassium ATP sensitive channels resulting in increased ROS in mitochondrial matrix and release of H2
in the cytoplasm [48
]. Further, antimycin A increases superoxide both in the matrix and extra mitochondrial space which can be trapped by mitochondrial scavengers and SOD1. Because the substrate deprivation is only for a short period of time (4 h), there are residual substrates available for mitochondria to use and programmed cell death can occur as energy dependant process. Alternatively, cells could die from energy independent mechanisms such as necrosis. Thus, the above processes (ATP depletion and antimycin A) both would release ROS (O2
) into the cellular cytoplasm where they can be scavenged by SOD1 and catalase.
Initiation of the death program is achieved by a wide variety of stimuli, with the identification of gene products that positively or negatively modulate the progression of the cell suicide pathway [49
]. Both the intrinsic (mitochondrial) and extrinsic (receptor) mediated pathways of apoptosis lead to caspase activation. In its active form, caspase-3, the effector protease of apoptosis plays a role in the proteolytic cleavage of proteins, such as the cleavage of nuclear DNA repair enzyme poly (ADP-ribose) polymerase and inhibitor of caspase-activated DNase [50
]. There is increasing evidence indicating that ROS and reactive nitrogen species (RNS) contribute to mitochondrial dysfunction and signal the activation of caspase-3 and initiation of cell death [51
]. Our results demonstrated that active caspase-3 expression was increased more in LLC-EGFP than LLC-SOD1 cells in response to ATP depletion injury, suggesting that over-expression of SOD1 partially reduces the severity of caspase-3 activation. However, SOD1 over-expression did not completely block caspase-activation following ATP depletion-recovery. Since SOD1 is most effective in scavenging of cytosolic O2
it is likely that other ROS such as H2
and mitochondrial O2
also plays a significant role in caspase-3 activation in our model of injury. To partly address this issue, we treated wild type LLC-PK1
cells with a mitochondrial antioxidant, MitoTEMPO. These results indicated that low doses of MitoTEMPO were indeed effective in reducing ATP depletion mediated cytotoxicity and apoptosis. At the highest dose, however MitoTEMPO was ineffective against ATP depletion induced cytotoxicity. It is possible that at higher doses, MitoTEMPO exhibits non-specific effects. Further, this finding was consistent with previously published studies that other mitochondrially targeted antioxidants such as MitoQ can result in increased H2
production at higher doses[53
]. In this regard, it is possible that in addition to O2
, other free radicals such as H2
also play an important in ATP depletion induced cytotoxicity. Recent studies have suggested that hydroperoxide produced by SOD1 in mitochondrial intermembrane space controls cytochrome c
catalyzed peroxidation [54
], leading to the production of H2
. In fact, neonatal mice over-expressing SOD1 have more hydroperoxide accumulation following ischemia compared to wild type mice [55
] and menadione treatment increases DCF fluorescence (an indicator of hydroperoxide) and cell death in SOD1 transgenic neurons compared to wild type neurons [56
]. Appropriate concentration of catalase has been shown to be protective in menadione toxicity [57
] and VUB induced apoptosis in normal human keratinocytes [58
]. Concurrent with these reports, in our study, addition of PEG-catalase to ATP depleted LLC-EFP cells significantly decreased cytotoxicity and caspase-3 activation. When catalase was added to the ATP depleted LLC-SOD1 cells, there was a trend for lower cytotoxicity and apoptosis in SOD1 + catalase cells, although this was not statistically significant. In our protocol, we added PEG-catalase at the beginning of the ATP depletion phase and replenished it during the recovery phase. Thus, it is likely that we may have encountered some loss of catalase during the experiment. So, to elucidate the synergistic protective effects of catalase and SOD1 ATP depletion-recovery, in future studies, it may be necessary to co-transduce SOD1 and catalase together by lentiviral vector mediated techniques to continuously increase the activity of catalase in proximal tubular epithelial cells.
In conclusion, these studies show for the first time that lentiviral vector mediated SOD1 over-expression is able to partially attenuate ATP depletion mediated cytotoxicity and caspase-3 activation in proximal tubular epithelial cells. However, it is likely that multiple signaling molecules such as H2O2 are also involved in ATP depletion mediated injury. Future studies will thus utilize genetic techniques to manipulate SOD1 vs. SOD2 and catalase (over-expression and silencing) in vitro to dissect the apoptotic signaling pathways involved.