In sepsis, reactive oxygen species (ROS) production is increased. This process takes place mainly within the electron transport chain. ROS production is part of the pathophysiology of multiple organ failure in sepsis. Succinate yields Dihydroflavine-Adenine Dinucleotide (FADH2), which enters the chain through complex II, avoiding complex I, through which electrons are lost. The aim of this work is to determine if parenteral succinate reduces systemic ROS production and improves kidney function. Rats with cecal ligation and puncture were used as model of sepsis, and 4 groups were made: Control group; Succinate group, which only received parenteral succinate; Sepsis group; and Sepsis which received parenteral succinate. Systemic ROS are measured 24 hours after the procedure. Rats subjected to cecal puncture treated with succinate had less systemic ROS than Septic untreated rats (
Sepsis is a pathology whose incidence is increasing, with a mortality rate that, according to a recent report, can reach 40% [
Triggering mechanisms of organ failure appear to be multiple. Oxidative stress seems to be one of these triggering mechanisms. There are different definitions of oxidative stress, but the most common and descriptive one is the imbalance between the production of reactive oxygen species (ROS) and cellular antioxidant capacity, which can potentially damage cells and destroy tissue [
Reactive oxygen species are a group of molecules which include oxygen radicals, such as superoxide (O2⋅−), hydroxyl (OH⋅), peroxide (RO2⋅), and alkoxide (RO⋅), as well as nonradicals that are oxidizing agents or that quickly become radicals, such as hypochlorous acid (HOCl), singlet oxygen (1O2), and hydrogen peroxide (H2O2) [
Over the last few years, there have been a large number of studies that describe oxidative stress in patients with sepsis, with evidence of ROS production, related damage, and antioxidant depletion [
When antioxidant defenses are outnumbered, oxidative stress, which can significantly damage lipids, proteins, and nucleic acids, occurs both within the mitochondria and the rest of the cell [
During sepsis, most ROS are produced in the mitochondria. It is believed that this is due to the electron loss that takes place when they are passed from complex I to complex III of the mitochondrial electron transport chain. On the other hand, the uncoupling of the chain also takes place between complex I and complex II [
The objective of this study is to observe whether the administration of intraperitoneal succinate to rats subjected to cecal ligation and puncture reduces ROS production and improves sepsis-induced kidney failure.
Male Sprague Dawley rats of 200 grams of average weight adapted to 12 h light cycles for 7 days and fed
Four groups were formed: (1) Control group; (2) Succinate group, to which intraperitoneal succinate was administered 2 hours before initiating the surgery of groups 3 and 4 and 2 hours before the taking of the sample; (3) Cecal Puncture group, on which the procedure described in the following item was performed; (4) Cecal Puncture and Succinate group, to which succinate was administered 2 hours before the surgery and 2 hours before the taking of the sample. 24 hours passed between the surgery and the taking of the sample (Figure
Procedure flow chart. Surgery was made only in groups 3 and 4. Succinate was administered in groups 2 and 4.
The procedure was performed under sedation and anesthesia with 100 mg/kg of intraperitoneal Ketamine and 2.5 mg/kg of intraperitoneal Xylazine. In accordance with the technique described in literature [
Intraperitoneal succinate solution was administrated on groups 2 and 4, according to the flow chart specifications. 5 mmol/kg of intraperitoneal succinate 0.4 M solution was administered. Solution was prepared from succinic acid (Sigma Chemical Co.), neutralized with NaOH, and sterilized by filtration.
Intraperitoneal succinate solution was administered to rats of groups 2 and 4. Two hours later, cecal ligation and puncture was performed to rats of groups 3 and 4. 24 hours later, another administration of intraperitoneal succinate solution was performed to rats of groups 2 and 4. Two hours later, blood samples were taken, and rats were sacrificed after that (Figure
A blood sample was taken through cardiac puncture under anesthesia with 100 mg/kg of intraperitoneal Ketamine and 2.5 mg/kg of intraperitoneal Xylazine, and the animal was later euthanized. Blood was centrifuged in a dry tube at 3000 rpm (900g) for 5 minutes. Then, the serum was separated and frozen at −75°C until measurement.
The measurement of systemic ROS was taken using 2
Blood sample was taken as described above. The measurements were processed on a Vitros 5600 Ortho Clinical Diagnostics analytical platform, using the dry chemistry method. Average results are expressed in mg/dl.
The Statistix 7.0 program was used. The Student
DCFH average emissions were as follows: group 1 (0.0332 (SD = 0.008)); group 2 (0.0352 (SD = 0.011)); group 3 (0.0759 (SD = 0.037)); and group 4 (0.0598 (SD = 0.006)) (Figure
Serum ROS levels. DCFH emission levels. ANOVA test showed differences between groups (
Average serum creatinine levels were as follows: group1 (0.39 mg/dl (SD = 0.07)); group 2 (0.4 mg/dl (SD = 0.07)); group 3 (0.54 mg/dl (SD = 0.08)); and group 4 (0.6 mg/dl (SD = 0.2)) (shown in Figure
Serum creatinine levels. ANOVA test showed differences between groups (
On the other hand, there was no correlation in creatinine levels among the 4 groups (rho = 0.24;
Correlation between DCFH levels and creatinine. There was no correlation between creatinine levels and DCFH emission (rho = 0.24;
It can be seen in this study that systemic ROS levels were elevated in septic rats, and administering parenteral succinate reduced ROS production in septic animal. Also, succinate did not reduce creatinine levels. At last, creatinine levels did not correlate with DCFH levels.
This study is innovative, as systemic ROS, or serum ROS, were measured in sepsis. Literature describes the presence of ROS at the tissue level and associates tissue damage with the presence of these molecules. In other studies, we measured the presence of ROS in yeast in the presence of Menadione [
It is known that ROS at the tissue level damage proteins, cell membranes, and nucleic acids. This damage can lead to cell death via apoptosis, which has been described many times. This description is mainly at an experimental level in cell lines or experimental models and at the tissue level. In literature, the presence of systemic ROS in an animal model of sepsis is described, but they are measured with the TBARS technique, and they are also increased in the model, but their presence is not associated with organ failure markers [
This study is also innovative, as systemic ROS levels were reduced in septic rats with succinate, a Krebs cycle intermediate, with no changes in nonseptic rats. This could be due to the fact that the electron transport chain is not affected in nonseptic rats; the effects of the drug are not the same as those seen in septic rats.
Chouchani et al. [
In sepsis, other mechanisms take place in the mitochondria. Several studies showed that there is low activity in the electron transport chain, in different complex. Lorente et al. showed lower activity of complex IV in platelets of septic patients [
In sepsis, electron transport chain activity and mitochondrial respiration are decreased, shown by the lower levels in oxygen consumption and the decrease in ATP levels and ATP/ADP ratio [
Several attempts have been made to reduce oxidative stress in septic patients, most of them with inconclusive results [
All these results might show that succinate in sepsis can improve systemic ROS levels restoring the electron transport chain, but this does not improve renal function measured with creatinine levels. Also, there is no correlation between systemic ROS and creatinine levels, indicating that ROS production and kidney damage are not linked. At last, we showed [
In this study, no correlation between systemic ROS levels and creatinine was found. Sepsis is the most common cause of kidney failure in the ICU [
The question that needs to be solved is that do ROS cause tissue damage or just molecules that show that mitochondrial processes are not working correctly. More studies are needed to solve this question.
Parenteral succinate reduces systemic ROS levels, but it does not reduce serum creatinine levels. Further studies are needed to understand this drug’s mechanism of action.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This study was funded by the Universidad de Buenos Aires, RS (CS) No. 8456/2017.