Ischemia In Vivo Induces Cardiolipin Oxidation in Rat Kidney Mitochondria

Simple Summary

Mitochondrial cardiolipin is a unique phospholipid that plays a vital role in ATP synthesis. It has been observed that ischemia/reperfusion causes damage to cardiolipins in the heart or brain tissues; however, very little data has been found regarding kidney cardiolipins and related damage after ischemia/reperfusion. Oxidative stress plays a key role during reperfusion. However, even during ischemia, cardiolipins may be oxidized. Therefore, we aimed to evaluate cardiolipin oxidation during renal ischemia in vivo. Renal ischemia in vivo was induced in male Wistar rats for a 30–60-minute period; we then isolated kidney mitochondria, extracted the lipids, and analyzed cardiolipin by applying chromatographic and mass spectrometric methods. The results showed that after even 30 min of in vivo ischemia, the amounts of the dominant cardiolipin species decreased almost in half, and it further decreased when extending the ischemia time. Cardiolipin was oxidized with up to eight additional oxygen atoms, yielding eight different species with multiple isomeric forms. This shows that even after ischemia, cardiolipin levels are altered, and many cardiolipin oxidation products are produced, which may also potentially be modified into more harmful lipid signaling molecules that may induce more damage to mitochondria.

Abstract

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. We aimed to analyze the effect of in vivo ischemia on cardiolipin oxidation. Adult male Wistar rats were anesthetized; ir 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 tetralin oleoyl cardiolipin and its oxidation products. Chromatographic and mass spectrometric analysis revealed a 47%, 68%, and 74% decrease in tetralin oleoyl 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 6.9-fold increase in all oxidized cardiolipin forms. We present evidence that renal ischemia in vivo alone induces tetralin oleoyl cardiolipin oxidation and depletion in rat kidney mitochondria.

Keywords

cardiolipin; ischemia; oxidation; mitochondria; kidney; oxidative stress.

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Introduction

Cardiolipin (CL) is a unique phospholipid that is mainly found in mitochondrial membranes and bacterial plasma membranes [1]. In the mitochondria, most CL is located in the inner membrane (IMM), making up about 20% of total IMM phospholipid content [2]. The structure of this phospholipid is dimeric and includes four fatty acyl chains, which give rise to a variety of decadent acid combinations and cardiolipin molecular species. Even though many CL species are found in mammalian tissues, there is one species, tetralinoleyl-CL, that is the most abundant [3,4]. For example, this CL species comprises approximately 80% of all cardiolipins in the hearts of humans, rodents, and bovines. It has been observed that linoleic acid is the most abundant fatty acid in the cardiolipins that are found in the liver, kidney, muscle, and spleen tissues [5,6]. This unique structure of cardiolipin gives it special properties and functions within mitochondria. CL participates in mitochondrial membrane morphology, bioenergetics, metabolite transport, mitophagy, immune response, and apoptosis [7–10]. Perhaps one of the most important functions of cardiolipins is their interaction with the mitochondrial respiratory chain complexes—cardiolipin is essential for their stability and the optimal function of the oxidative phosphorylation system. Previous studies have shown that cardiolipin is necessary for the optimal functioning of complex I, complex II, complex III, cytochrome c, and adenosine triphosphate (ATP) synthase [10–15]. It has also been demonstrated that cardiolipin can increase the efficiency of oxidative phosphorylation by at least 35% [16]. Thus, any damage to cardiolipin may cause mitochondrial dysfunction and further pathological cascades. Changes in cardiolipin structure and amounts have been observed during such pathologies as Barth syndrome, cardiovascular and neurodegenerative diseases, and myocardial ischemia/reperfusion [3,17–20].

Structural changes in cardiolipin can occur mainly during oxidative stress when cardiolipin is oxidized through the attack of reactive oxygen species (ROS). Mitochondria are considered a significant source of ROS production; therefore, considering the location of cardiolipin as well as its high number of polyunsaturated fatty acids, CL becomes highly susceptible to oxidation [21,22]. Under physiological conditions, ROS production and scavenging are highly controlled; however, it has been observed that ROS production increases under various pathological conditions such as ischemia/reperfusion, aging, and degenerative diseases [23]. It has been shown that myocardial ischemia/reperfusion causes significant loss of cardiolipin in rat heart mitochondria and reduced activity in I, III, and IV electron transport chain complexes [11–13]. Short periods of ischemia might not alter the antioxidant system; however, prolonged ischemia (e.g., 60 min of heart ischemia) damages the electron transport chain and increases ROS generation even when 1,5% oxygen is left [23,24]. Increased ROS generation during ischemia can already induce the oxidation of polyunsaturated fatty acids in CL with the formation of lipid peroxides and cause further damage to mitochondria.

Much research has been done analyzing cardiolipin alterations after myocardial ischemia/reperfusion [18,19,25,26]. However, there is not enough information about the implications of kidney ischemia/reperfusion on cardiolipin in kidney mitochondria. As there are no data about cardiolipin alterations during kidney ischemia at all, this study aimed to evaluate qualitative and quantitative changes of cardiolipin in kidney mitochondria after warm in vivo ischemia. This study demonstrates that kidney ischemia in vivo causes peroxidation of CL. Several molecular species of cardiolipin containing up to eight additional oxygen atoms attached to polyunsaturated fatty acids were identified using the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method.

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Materials and Methods

1. Animals and Experimental Ischemia In Vivo Model

The experimental procedures used in the present study were performed according to the Lithuanian Committee of Good Laboratory Animal Use Practice (No. G2-170, 2021-03- 02). Adult male Wistar rats weighing 200–250 g were housed under standard laboratory conditions, maintained on a natural light and dark cycle, and had free access to food and water ad libitum. Animals were acclimatized to laboratory conditions before the experiment. Pentobarbital and ketamine were used to perform anesthesia. Vascular clips were placed over the rats’ renal arteries to induce renal ischemia (37 ◦C) in vivo. Ischemia was confirmed by observing the kidneys change color from red to purple within ~2 min. At the end of ischemia (30 min, 40 min, or 60 min), the clips were taken off, and the kidneys were removed and washed free of blood in a cold (0–4 ◦C) 0.9% KCl solution. After that, kidney tissue was used for the isolation of mitochondria. Sham-operated rats (control group) underwent identical surgical procedures except for applying vascular clips.

2. Isolation of Kidney Mitochondria

Kidney tissue was cut into small pieces and homogenized in an isolation medium containing 250 mM sucrose, 10 mM Tris HCl, and 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.3). Cytosolic and mitochondrial fractions were separated by differential centrifugation (5 min at 750× g and 10 min at 10,000× g, two times), and the pellet was suspended in an isolation medium. The pellet was used for protein determination by the Biuret method, mitochondrial function determination, and phospholipid extraction.

3. Determination of Mitochondrial Function

Mitochondrial function was determined to confirm ischemic damage to mitochondria. This was done by measuring mitochondrial respiration (oxygen consumption) with the Oroboros-2k polygraph system in a 2 mL incubation medium (150 mM KCl, 10 mM Tris·Cl, 5 mM KH2PO4, 1 mM MgCl2 × 6H2O) at pH 7.3 and 37 ◦C. State II (V0) respiration was measured in the presence of 5 mM glutamate and 5 mM malate as complex I-dependent substrates. State III (V3) respiration was measured by adding 2 mM adenosine diphosphate (ADP). Mitochondrial outer membrane permeability was assessed by adding 32 µM cytochrome c (V3+cytc). The respiration was inhibited by adding 0.12 mM carboxyatractyloside (Vcat), and changes in respiration rates were assessed, which indicated alterations in mitochondrial inner membrane permeability. Real-time data acquisition and data analysis were performed with Datlab 5 software (Oroboros Instruments, Innsbruck, Austria). Oxygen consumption rates were expressed as pmol/s/0.25 mg mitochondrial protein. Original curves of mitochondrial respiration in control and ischemic (30, 40, 60 min) groups are represented in Figure 1.

Figure 1. Oxygen consumption in kidney mitochondria in control and ischemia groups. In the curves, the blue trace represents oxygen concentration (nmol/mL), and the red trace represents oxygen flux (pmol/s/0.25 mg mitochondrial protein). Vg: respiration rate in the presence of 0.5 mg/mL of mitochondria (mix) and substrates, 5 mM glutamate, and 5 mM malate; V3: maximal respiration rate after adding 2 mM ADP; V3+cyte: respiration rate after adding 32 uM cytochrome c; Vcat: respiration rate after adding 0.12 mM carboxyl tractyloside. It was observed that mitochondrial respiration was diminished in all ischemia groups compared to control.

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4. Extraction of Phospholipids from Kidney Mitochondria

Phospholipid extraction from kidney mitochondria was carried out using a modified Folch’s method [27]. A total of 500 µL of mitochondrial suspension was mixed with 1 mL of deionized water, 2 mL of methanol, and 4 mL of chloroform (1:2:4 vol.). The mixture was centrifuged at room temperature at 2000× g speed for five minutes. The lower chloroform phase–containing lipids–was recovered and evaporated under a N2 stream in a glass vial. The remaining upper phase was mixed with an additional 2 mL of chloroform and centrifuged again. After that, the lower phase was recovered and added to the glass vial. This last step was repeated two times to recover as much lipid content as possible from the mixture. After concentration under an N2 stream, the pellet was resuspended in 100 µL of chloroform and 500 µL isopropanol (1:5 vol.) and filtered through a 45 µm syringe filter before the analysis.

5. Oxidation of Cardiolipin Standard

Cardiolipin standard (cardiolipin sodium salt from bovine heart, >97% (TLC))lyophilized powder, Sigma Aldrich, St. Louis, MO, USA) was oxidized using a method based on that reported by Kagan and Tyurina (28,29]. Cardiolipin standard was dissolved in phosphate buffer (K2HPO4 50 mM, KHPO4 50 mM, double-distilled H2O; pH 7.4) and vortexed. The mixture was placed in an ultrasonic bath for 3 min to form lipid vesiclesThe mixture was treated with cytochrome c (30 uM) and HO, (400 uM) for one hour at 37 C in a water bath. HO was added every 15 min (a total of four times) during the incubation period. Lipids were then extracted in chloroform/methanol solvent using a method described earlier.

6. Separation and Evaluation of Cardiolipin

Cardiolipin separation and analysis from our standard and mitochondrial samples were performed by ultra-effective liquid chromatography—mass spectrometry (UPLC-MS) using a Waters Acquity UPLC chromatograph coupled with a Xevo TQD (triple quadrupole) mass spectrometer with electrospray ionization (ESI). For UPLC analysis, an Acquity UPLC BEH C18 1.7 µm column was used. The method that was used was based on the reverse-phase ion-pair high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method that was described by Kim et al. [30]. Gradient elution used two eluents: eluent A (450 mL acetonitrile, 50 mL water, 2.5 mL triethylamine, 2.5 mL glacial acetic acid) and eluent B (450 mL isopropanol, 50 mL water, 2.5 mL triethylamine, 2.5 mL glacial acetic acid). The elution gradient is shown in Table 1.

7. Statistical Analysis

The quantitative data are presented as means ± standard error. Statistical analysis and graphic visualization were performed using the SigmaPlot v14.0 software package (Systat Software Inc., Chicago, IL, USA). For the comparison of group means, one-way ANOVA was applied, followed by Dunn’s post hoc test for the pairwise multiple comparison procedure. Additionally, Student’s/Welch’s t-test was used for each pair mean comparison if the Shapiro–Wilk normality test was passed; Mann–Whitney Sum Rank test was used otherwise. p values < 0.05 were considered statistically significant.

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Discussion

Ischemia and reperfusion remain a serious clinical problem that damages the affected organs and tissues. The molecular mechanism of this injury involves increased reactive oxygen species generation in mitochondria and cardiolipin peroxidation and depletion, which in turn impairs mitochondrial oxidative phosphorylation and mitochondrial and cellular integrity [21,32,33]. Mitochondrial damage already occurs during ischemia, and although the level of oxygen is reduced, reactive oxygen species are still generated at a level that is enough to oxidize and deplete cardiolipin [34]. Cardiolipin peroxidation during ischemia-reperfusion has mostly been analyzed in heart mitochondria; however, there is a lack of research that has been done on cardiolipin in renal mitochondria.

This study shows for the first time that tetralin oleoyl cardiolipin is oxidatively modified and depleted in rat kidney mitochondria during in vivo kidney ischemia. In the first stage, a standard tetralin oleoyl cardiolipin was used for in vitro oxidation using cytochrome c and hydrogen peroxide for the analysis of cardiolipin oxidation products. Since cardiolipin closely interacts with cytochrome c in the mitochondria, cardiolipin can be readily oxidized by cytochrome c peroxidase [35]. Additionally, Aluri et al. showed that during mouse heart ischemia and reperfusion, the inhibition of the distal part of the electron transport chain, mainly the IV complex, evokes cytochrome c peroxidase activity, which, together with hydrogen peroxide, can readily oxidize cardiolipin [36]. Using UPLC-MS, eight cardiolipin oxidation products that had from one to eight additional oxygen atoms attached to linoleic acids were evaluated. Based on mass spectrometry results, up to four oxygen atoms could be found on a single linoleic acid in the form of hydroxides or hydroperoxides. Similar findings were also published by Kim et al.; however, different oxidizing agents were used, specifically air/2,2’-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH)/photosensitizer, and light. Furthermore, similar findings were also published by Helmer et al., who applied a Fenton reaction. The authors described in detail cardiolipin oxidation products with additional two and four oxygen atoms. Even though Helmer et al. showed that up to eight oxygen atoms were detected on cardiolipin, no further details on the rest of the cardiolipin forms were provided [30,37]. The ion fragmentation patterns were similar to those described in this study, which may show that cardiolipin oxidation with cytochrome c and hydrogen peroxide follow the same patterns of oxidation. Among the oxidation products of standard cardiolipin, the dominating form was cardiolipin with two additional oxygen atoms—cardiolipin hydroperoxide, which was also observed by Helmer et al. [37]. However, our study shows that in the in vivo setting, the relative amounts of oxidized cardiolipin forms might differ since the most abundant forms were cardiolipin with an additional five and four oxygen atoms in control and ischemic rat kidney mitochondria. This indicates that cardiolipin hydroperoxide might still be reactive and undergo further oxidation in vivo, producing somewhat more stable oxidized cardiolipin forms.

In the next set of experiments, we analyzed the quantitative and qualitative changes of rat kidney cardiolipin after 30–60 min in vivo ischemia. Firstly, it was determined that the levels of tetralin oleoyl cardiolipin and trilinoleoyl-monooleoyl cardiolipin are almost the same in rat kidney mitochondria, which makes both of them dominant. After 30 min of in vivo ischemia, the levels of both cardiolipin species decrease in half; however, only after 30 min and 40 min ischemia is the decrease significant for tetralin oleoyl cardiolipin. Petrosillo et al. analyzed cardiolipin peroxidation in ischemic rat hearts and demonstrated that 30 min of ischemia caused a decrease in cardiolipin content by 28% [33]. Similar results were obtained by Lesnefsky et al. while analyzing rabbit heart mitochondria in ischemic conditions. A total of 30 min ischemia caused a slight decrease in cardiolipin and cytochrome c content in subsarcolemmal mitochondria; however, no change in cardiolipin or cytochrome c content was observed in interfibrillar mitochondria [25]. Chen and Lesnefsky further investigated this model of ischemia and found that after 30 and 45 min of ischemia, the production of hydrogen peroxide significantly increased, which means that cardiolipin can be readily oxidated in ischemic conditions [38]. It has also been shown that even 20 min of warm renal ischemia in vitro caused significant damage to the mitochondrial oxidative phosphorylation system as well as an increase in proapoptotic factor activity [39]. However, these experiments were done ex vivo/in vitro, and so far, not many in vivo experiments have been done to provide any information about cardiolipin alterations during ischemia alone, especially in kidneys. Kim et al. analyzed cardiolipin content in rat brain and heart after asphyxia-induced cardiac arrest and discovered that tetralin oleoyl cardiolipin is only slightly depleted in rat heart, but not the brain, after 30 min of cardiac arrest [40]. Liu et al. induced 45 min of rat kidney ischemia in vivo and discovered that kidney capillary endothelial cell mitochondria became swollen with a loss of cristae membranes which was attenuated by a cardiolipin-targeted peptide SS-31. Even though the authors did not analyze cardiolipin, their results indicate that the loss of cardiolipin could have induced morphological changes in mitochondria [41].

Not much is known about cardiolipin oxidation products during ischemia, especially in ischemia in vivo. This study shows that rat kidney ischemia in vivo causes tetralin oleoyl cardiolipin oxidation with up to eight additional oxygen atoms, forming eight oxidation products. It was shown, by Lesnefsky et al., that 25 min of aged rat heart ischemia caused a significant increase in 1496 Dalton cardiolipin (with an additional three oxygen atoms) in subsarcolemmal and interfibrillar mitochondria. Interestingly, the amount of this cardiolipin species in rat heart mitochondria after 25 min of ischemia was higher compared to 25 min of ischemia followed by 30 min of reperfusion [26]. Our study showed that this oxidized cardiolipin species significantly increases after 30 min of in vivo rat kidney ischemia; however, it decreases almost to control levels after 60 min of ischemia. Such a tendency was noticed with other cardiolipin oxides that had an extra four, five, six, seven, and eight oxygen atoms. We hypothesize that longer ischemia times might trigger oxidized cardiolipin hydrolysis by phospholipases into various lysocardiolipin species and linoleic acid peroxides [42,43]. Indeed, Smaalen et al. have used a porcine model of clinical kidney transplantation design with sub normothermic (28 ◦C) ischemic perfusion and showed that kidney ischemia in pigs causes significant elevation of monolysocardiolipin (trilinoleoyl cardiolipin) [44]. Such monolysocardiolipins are no longer suitable for the induction of mitochondrial inner membrane curvature, nor do they associate with mitochondrial proteins or native cardiolipin, which causes a decrease in the activity of oxidative phosphorylation and release of cytochrome c [45]. Paradies et al. have shown that loss of cardiolipin caused a decrease in the activity of complexes I, III, and IV, which was restored with exogenous cardiolipin, but not with monolysocardiolipin or oxidated cardiolipin [11–13].

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Conclusions

This study shows that kidney ischemia in vivo alone causes severe cardiolipin oxidation and depletion in rat kidney mitochondria. Further studies are needed to assess possible metabolites (like monolysocardiolipin, free linoleic acid peroxides, and 4-hydroxynonenal) that can come out of the catabolism of oxidized cardiolipin and participate in cell signaling that may lead to renal cell death. Additionally, the prevention of cardiolipin oxidation and depletion during ischemia could protect kidneys from the increased damage that occurs during reperfusion.

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Arvydas Strazdauskas 1,2, Sonata Trumbeckaite 1,3, Valdas Jakstas 3,4 , Justina Kamarauskaite 3,4 , Liudas Ivanauskas 5 and Rasa Baniene 1,2

1 Laboratory of Biochemistry, Neuroscience Institute, Lithuanian University of Health Sciences, LT-50162 Kaunas, Lithuania; sonata.trumbeckaite@lsmuni.lt (S.T.); rasa.baniene@lsmuni.lt (R.B.)

2 Department of Biochemistry, Medical Academy, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania

3 Department of Pharmacognosy, Medical Academy, Lithuanian University of Health Sciences, LT-50162 Kaunas, Lithuania; valdas.jakstas@lsmuni.lt (V.J.); justina.kamarauskaite@lsmu.lt (J.K.)

4 Laboratory of Biopharmaceutical Research, Institute of Pharmaceutical Technologies, Lithuanian University of Health Sciences, LT-50162 Kaunas, Lithuania

5 Department of Analytical and Toxicological Chemistry, Medical Academy, Lithuanian University of Health Sciences, LT-50162 Kaunas, Lithuania; liudas.ivanauskas@lsmuni.lt