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. 2020 Oct 13:2020:8855585.
doi: 10.1155/2020/8855585. eCollection 2020.

Increased Succinate Accumulation Induces ROS Generation in In Vivo Ischemia/Reperfusion-Affected Rat Kidney Mitochondria

Justina Kamarauskaite  1   2 , Rasa Baniene  1   3 , Darius Trumbeckas  4 , Arvydas Strazdauskas  1   3 , Sonata Trumbeckaite  1   2
Affiliations

Increased Succinate Accumulation Induces ROS Generation in In Vivo Ischemia/Reperfusion-Affected Rat Kidney Mitochondria

Justina Kamarauskaite et al. Biomed Res Int. .

Abstract

Mitochondria are recognized as main reactive oxygen species (ROS) producers, involving ROS generation by mitochondrial complexes I and III. Lately, the focus has been shifting to the ROS generation by complex II. Contribution of complex II (SDH) to ROS generation still remains debatable, especially in in vivo settings. Moreover, it is not completely defined at what time of ischemia the first alterations in mitochondria and the cell begin, which is especially important with renal arterial clamping in vivo during kidney surgery, as it predicts the postischemic kidney function. The aim of this study on an in vivo rat kidney ischemia/reperfusion model was to determine if there is a connection among (a) duration of kidney ischemia and mitochondrial dysfunction and (b) succinate dehydrogenase activity, succinate accumulation, and ROS generation in mitochondria at low and saturating succinate concentrations. Our results point out that (1) mitochondrial disturbances can occur even after 30 min of kidney ischemia/reperfusion in vivo and increase progressively with the prolonged time of ischemia; (2) accumulation of succinate in cytosol after ischemia/reperfusion correlated with increased H2O2 generation mediated by complex II, which was most noticeable with physiological succinate concentrations; and (3) ischemia/reperfusion induced cell necrosis, indicated by the changes in LDH activity. In conclusion, our new findings on the accumulation of succinate in cytosol and changes in SDH activity during kidney ischemia/reperfusion may be important for energy production after reperfusion, when complex I activity is suppressed. On the other hand, an increased activity of succinate dehydrogenase is associated with the increased ROS generation, especially with physiological succinate concentrations. All these observations play an important role in understanding the mechanisms which occur in the early phase of ischemia/reperfusion injury in vivo and may provide new ideas for novel therapeutic approaches or injury prevention; therefore, more detailed studies are necessary in the future.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Original curves of kidney mitochondrial respiration in the control and ischemia/reperfusion (I/R) groups. Mitochondrial respiration rate was measured as described in Methods using 5 mM glutamate plus 5 mM malate as substrates. The blue trace represents oxygen concentration (nmol/mL), and the red trace represents oxygen flux. I20/R30, I30/R30, I40/R30, and I60/R30 refer to different ischemia durations (respectively, 20, 30, 40, and 60 minutes) following by 30-minute reperfusion. V0: routine respiration rate in the presence of 0.5 mg/mL of mitochondria and substrates; V3: state 3 respiration rate in the presence of 1 mM ADP; V3+cyt c: state 3 respiration rate in the presence of 32 μM cytochrome c; VATR (+cyt c): respiration rate in the presence of 0.12 mM atractyloside and 32 μM cytochrome c.
Figure 2
Figure 2
Effect of ischemia on complex I (a), complex II+III (b), and succinate dehydrogenase (c) activity in kidney mitochondria. The complex I activity was measured spectrophotometrically at 340 nm as described in Methods. ∗p < 0.05vs. control. The complex II activity was measured spectrophotometrically at 550 nm as described in Methods. ∗p < 0.05vs. control. The SDH activity was measured spectrophotometrically at 600 nm as described in Methods. ∗p < 0.05vs. control.
Figure 3
Figure 3
Effect of ischemia on H2O2 generation in kidney mitochondria with glutamate/malate (a) and succinate: saturating (b) and unsaturating (c) concentrations. The generation of H2O2 in kidney mitochondria was determined fluorimetrically as described in Methods (excitation at 544 nm, emission at 590 nm). Substrates: (a) glutamate (5 mM) + malate (5 mM); (b) succinate 5 mM; (c) succinate 0.4 mM. Respiratory chain complex inhibitors: rotenone (5 μM) and myxothiazol (2 μM). ∗p < 0.05 vs. succinate 5 mM group; #p < 0.05 vs. control group; &p < 0.05 vs. succinate 5 mM group; λp < 0.05 vs. control group; $p < 0.05 vs. succinate 0.4 mM group; ϕp < 0.05 vs. succinate 5 mM, with rotenone group; §p < 0.05 vs. succinate 5 mM, with rotenone and myxothiazol group; ψp < 0.05 vs. control group; κp < 0.05 vs. succinate 0.4 mM, with rotenone group; αp < 0.05 vs. succinate 5 mM, with rotenone and myxothiazol group.
Figure 4
Figure 4
Effect of kidney ischemia on LDH activity in cytosolic fraction. LDH activity was measured as described in Methods. ∗p < 0.05vs. control.
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