In Vitro Hypoxia/Reoxygenation Induces Mitochondrial Cardiolipin Remodeling in Human Kidney Cells.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

Ischemia In Vivo Induces Cardiolipin Oxidation in Rat Kidney Mitochondria.

Cardiolipin is a mitochondrial phospholipid that plays a significant role in mitochondrial bioenergetics. Cardiolipin is oxidized under conditions like oxidative stress that occurs during ischemia/reperfusion; however, it is known that even during ischemia, many reactive oxygen species are generated. Our aim was to analyze the effect of in vivo ischemia on cardiolipin oxidation. Adult male Wistar rats were anesthetized; then, their abdomens were opened, and microvascular clips were placed on renal arteries for 30, 40 or 60 min, causing ischemia. After ischemia, kidneys were harvested, mitochondria were isolated, and lipids were extracted for chromatographic and mass spectrometric analysis of tetralinoleoyl cardiolipin and its oxidation products. Chromatographic and mass spectrometric analysis revealed a 47%, 68% and 74% decrease in tetralinoleoyl cardiolipin after 30 min, 40 min and 60 min of renal ischemia, respectively (p < 0.05). Eight different cardiolipin oxidation products with up to eight additional oxygens were identified in rat kidney mitochondria. A total of 40 min of ischemia caused an average of a 6.9-fold increase in all oxidized cardiolipin forms. We present evidence that renal ischemia in vivo alone induces tetralinoleoyl cardiolipin oxidation and depletion in rat kidney mitochondria.

Caffeic Acid Phenethyl Ester Protects Kidney Mitochondria against Ischemia/Reperfusion Induced Injury in an In Vivo Rat Model.

To improve ischemia/reperfusion tolerance, a lot of attention has been focused on natural antioxidants. Caffeic acid phenethyl ester (CAPE), an active component of the resinous exudates of the buds and young leaves of Populus nigra L., Baccharis sarothroides A., etc., and of propolis, possesses unique biological activities such as anti-inflammatory, antioxidant, immunomodulating, and cardioprotective effects, among others. There is a lack of studies showing a link between the antioxidant potential of CAPE and the mechanism of protective action of CAPE at the level of mitochondria, which produces the main energy for the basic functions of the cell. In the kidney, ischemia/reperfusion injury contributes to rapid kidney dysfunction and high mortality rates, and the search for biologically active protective compounds remains very actual. Therefore, the aim of this study was to identify the antioxidant potential of CAPE and to investigate whether CAPE can protect rat kidney mitochondria from in vivo kidney ischemia/reperfusion induced injury. We found that CAPE (1) possesses antioxidant activity (the reducing properties of CAPE are more pronounced than its antiradical properties); CAPE effectively reduces cytochrome c; (2) protects glutamate/malate oxidation and Complex I activity; (3) preserves the mitochondrial outer membrane from damage and from the release of cytochrome c; (4) inhibits reactive oxygen species (ROS) generation in the Complex II (SDH) F site; (5) diminishes ischemia/reperfusion-induced LDH release and protects from necrotic cell death; and (6) has no protective effects on succinate oxidation and on Complex II +III activity, but partially protects Complex II (SDH) from ischemia/reperfusion-induced damage. In summary, our study shows that caffeic acid phenethyl ester protects kidney mitochondrial oxidative phosphorylation and decreases ROS generation at Complex II in an in vivo ischemia/reperfusion model, and shows potential as a therapeutic agent for the development of pharmaceutical preparations against oxidative stress-related diseases.

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

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.