Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1985-1998 © The Author(s) 2025. This article is an open access publication.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 12, pp. 2093-2107.

1985

Succinate Confers Stronger Cytoprotection

in Kidney Cells than in Astrocytes Due

to Its More Efficient Involvement in Energy Metabolism

Marina I. Buyan

1,2

, Kseniia S. Cherkesova

1,3

, Anna A. Brezgunova

Irina B. Pevzner

, Nadezda V. Andrianova

, and Egor Y. Plotnikov

1,a

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

e-mail: plotnikov@belozersky.msu.ru

Received August 18, 2025

Revised October 30, 2025

Accepted November 3, 2025

Abstract— Being among the most metabolically active organs, brain and kidneys critically depend on efficient

energy metabolism, which primarily relies on oxidative phosphorylation. Acute pathological conditions asso-

ciated with a lack of metabolic substrates or their impaired utilization trigger signaling cascades that initiate

cell death and lead to poorly reversible organ dysfunction. One of the therapeutic approaches to correct the

energy deficit is administration of exogenous metabolites of the tricarboxylic acid cycle, such as succinate.

Inthis study, we investigated the effects of exogenous succinate on astrocytes and renal epithelial cells under

normal conditions and in serum deprivation-induced injury. Incubation with succinate increased the viability

of both cell types under normal and pathological conditions, but a more pronounced cytoprotective effect was

observed in renal cells. In injured renal epithelial cells, succinate increased mitochondrial membrane poten-

tial, a critical parameter for the maintenance of mitochondrial function and ATP generation. Comparison of

respiration and oxidative phosphorylation parameters in astrocytes and renal epithelial cells in the presence

of exogenous succinate revealed that epithelial cells exhibited a significantly higher respiratory control and

lower proton leak compared to astrocytes, which correlated with the higher cytoprotective activity of succi-

nate for kidney cells. Therefore, succinate showed a noticeable positive effect in the renal epithelium both

under normal conditions and after serum deprivation; however, in astrocytes, its effect was less pronounced.

This discrepancy might be related to a more efficient succinate utilization by the mitochondria in renal

cells and intrinsic bioenergetic differences between astrocytes and epithelial cells. Despite the clinical use of

succinate-containing drugs, the determination of optimal dosages and development of effective therapeutic

regimens require further investigation. Our results demonstrate cell type-dependent differences in the efficacy

of succinate, suggesting that its therapeutic potential may differ significantly depending on the organ-specific

bioenergetic and metabolic properties.

DOI: 10.1134/S0006297925602539

Keywords: brain, astrocytes, kidneys, apoptosis, energy substrates, succinate, mitochondria, oxidative phos-

phorylation

* To whom correspondence should be addressed.

INTRODUCTION

Severe pathological conditions, such as ischemic

disease (including myocardial infarction and ischemic

stroke), various forms of shock (septic, hypovolemic,

and others), and acute and chronic heart failure in

the decompensation stage provoke systemic and lo-

cal tissue hypoperfusion, resulting in strong depriva-

tion of key energy substrates in organs and tissues.

The ensuing energy crisis triggers pathophysiological

BUYAN et al.1986

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

cascades, the central event being dysregulation of

cellular energy metabolism. This disrupts the balance

between anabolic and catabolic processes and sup-

presses key metabolic and signaling pathways [1, 2],

leading to cell death. The functioning of organs with

a high basal energy demand, such as the brain, heart,

and kidneys, critically depends on a high rate of ad-

enosine triphosphate (ATP) synthesis, which makes

them particularly vulnerable to such disruptions in

metabolism. The kidneys are among the most met-

abolically active human organs [3, 4] due to a high

density of mitochondria in the tubular epithelial cells

[5, 6]. This ultrastructural feature is directly related

to the significant energy expenditure required for

ATP-dependent processes, primarily, active tubular re-

absorption of electrolytes, glucose, amino acids, and

water. The energy expenditure of the brain is also

very high. Despite its relatively small mass, the brain

consumes approximately 20% of all oxygen and 25%

of glucose utilized by the human body at rest [7, 8].

High metabolic demands are characteristic not only

of neurons, but astrocytes as well. Astrocytes play a

key role in the neurometabolic interactions; they ac-

tively capture glucose from the bloodstream, metabo-

lize it glycolytically to lactate, and supply this lactate

to neurons via the astrocyte-neuron lactate shuttle

as an energy substrate for oxidative phosphorylation

in the mitochondria [8-10]. Hence, astrocytes require

metabolic substrates both for their own needs and to

maintain the energy level in neurons.

A decreased supply of nutrients to tissues leads

to impaired mitochondrial function, in particular, in-

efficient oxidative phosphorylation. The damage to

the mitochondria not only reduces ATP production

but also induces oxidative stress, ultimately resulting

in cell death [2,  11]. It is commonly accepted that

the use of additional energy substrates can promote

metabolic adaptation and prevent energy collapse

by enabling rapid metabolism of tricarboxylic acid

(TCA) cycle intermediates and replenishment of re-

ducing equivalents utilized in the electron transport

chain [12]. Since mitochondrial dysfunction caused

by cell damage leads to extensive cell death, resto-

ration of normal mitochondrial function and mainte-

nance of oxidative phosphorylation and ATP synthesis

are considered promising therapeutic approaches in

acute conditions [13]. Succinic acid salts have long

been proposed as therapeutic agents for this purpose.

However, no systematic in  vitro studies of the effects

of succinate salts on the bioenergetic processes in

various cell types under physiological and stress con-

ditions have yet been fully conducted.

Here, we investigated the efficacy of disodium

succinate in preventing the death of astrocytes and

renal epithelial cells in the invitro model of cellular

stress induced by serum deprivation (a classic method

for inducing cellular damage). We also analyzed its

effects on the key parameters of mitochondrial func-

tion, including transmembrane potential and cellular

respiration. The aim of this study was to compare

the molecular mechanisms of succinate-mediated cy-

toprotection in astrocytes and renal tubular epithelial

cells under physiological and stress conditions, with

a focus on the mitochondrial function as a potential

basis for this protective effect.

MATERIALS AND METHODS

Cell cultures. Experiments were performed in

primary astrocyte cultures isolated from the brains

of neonatal rats and in cultured normal rat kidney

epithelial NRK-52E cells (CRL-1571; ATCC, USA). All

animal procedures were carried out in accordance

with the guidelines of the Federation of European

Laboratory Animal Science Associations (FELASA)

and the ARRIVE guidelines. Animal protocols were

approved by the Ethics Committee of the Belozer-

sky Institute of Physico-Chemical Biology, Lomonosov

Moscow State University (protocol 006-1/2/2024). For

the experiments, neonatal rats were humanely euth-

anized with CO

. The skull was then opened asepti-

cally, the brain hemispheres were removed, and the

meninges were excised. Brain tissue was transferred

to 0.05% trypsin-EDTA solution (BioinnLabs, Russia),

incubated for 30min, washed with trypsin-EDTA solu-

tion, and resuspended in complete culture medium

containing DMEM with 1  g/L glucose (BioinnLabs)

and F-12 (BioinnLabs) at a 1  :  1 ratio, supplemented

with stable L-glutamine (BioinnLabs) and 10% fetal

bovine serum (FBS) (HiMedia, India). The suspension

was transferred to a culture flask pre-coated with

poly-D-lysine (Sigma-Aldrich, USA). After cells formed

a monolayer, they were washed to remove the mi-

croglia by shaking at 135  rpm for 16  h. The medium

was then removed, and astrocytes were detached

with 0.05% trypsin-EDTA solution to be used in the

experiments. NRK-52E cells were cultured in a medi-

um containing F-12 and DMEM at a 1  :  1 ratio with

1  g/L glucose and stable L-glutamine supplemented

with 10%  FBS (HiMedia). The cells were passaged

using 0.05%  trypsin-EDTA solution. Both astrocytes

and renal epithelial cells were incubated until the

formation of 50% monolayer, after which the control

cells received normal culture medium, while experi-

mental cells were grown in the medium containing

disodium succinate (Sigma-Aldrich). Serum depriva-

tion was performed by incubating the cells in the

medium containing all components except FBS (with

or without succinate) for 48  h, after which the cells

were analyzed. For all treatments, medium pH was

monitored using phenol red.

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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Cell viability assay. Astrocytes and renal epithe-

lial cells were seeded in 96-well plates, cultured until

50% confluence, and treated with disodium succinate

(12.5, 25, 50, and 100  mM) under normal and serum

deprivation conditions. Cell viability was assessed us-

ing an MTT assay (Macklin, China). The MTT reagent

was dissolved at 5  mg/mL in DMEM/F-12 medium

without sodium bicarbonate (GE Healthcare, USA) and

incubated with cells for 1  h at 37°C. The cells were

then lysed with dimethyl sulfoxide (Macklin, China),

and the optical density of formazan was measured at

595 nm with a Zenyth 3100 multimodal plate reader

(Anthos Labtec, Austria). Cells incubated with water

for 24 h served as a negative control.

Cell death assessment. To study the effect of

succinate on cell death, the cells were stained with

propidium iodide (PI) according to the manufactur-

er’s instructions (Lumiprobe, Russia). To visualize

live cells, the cells were stained with the vital dye

Calcein  AM as recommended by the manufacturer

(Invitrogen, USA) and imaged using an LSM  900 con-

focal microscope (Carl Zeiss, Germany) at the exci-

tation wavelengths of 543  nm for PI and 488  nm for

Calcein  AM, with emission recorded at 560-590  nm

for PI and 505-530  nm for Calcein  AM.

Mitochondrial transmembrane potential mea-

surement. To assess changes in the mitochondrial

membrane potential, astrocytes and kidney epitheli-

al cells were incubated with 50  mM disodium succi-

nate for 48 h and stained with 200  nM tetramethyl-

rhodamine ethyl ester (TMRE, Invitrogen) for 20  min

at 37°C. TMRE-stained cells were visualized with

LSM 900 microscope at the excitation wavelength of

543  nm with emission detected at 616-700  nm. Subse-

quent image processing was performed with the Fiji/

ImageJ software by analyzing the average TMRE flu-

orescence intensity in the cells.

Cellular respiration analysis. The oxygen con-

sumption rate (OCR) of astrocytes and kidney epithe-

lial cells was measured using a Seahorse XFp analyz-

er (Seahorse Bioscience, USA). The cells were seeded

in the wells of a Seahorse XF8 microplate. The fol-

lowing day before the experiment, the medium in the

wells was replaced with a Seahorse XF DMEM base

medium (Agilent, USA) supplemented with 2  mM glu-

tamine (BioinnLabs), and the cells were incubated for

one hour at 37°C. The plate was then placed in the an-

alyzer, and various stressors (all from Sigma-Aldrich)

were added to the indicated final concentrations in

the following order: 1)  disodium succinate, 50  mM;

2)  oligomycin, 4.5  μM; 3)  carbonyl cyanide-p-trifluo-

romethoxyphenylhydrazone (FCCP), 1  μM; 4)  a com-

bination of rotenone (2.5  μM), antimycin  A (4  μM),

and 2-deoxy-D-glucose (2-DG, 50  mM). In some experi-

ments, the substances were added in a different order:

1)  rotenone, 2.5  μM; 2)  disodium succinate, 50  mM;

3)  antimycin  A, 4  μM. The obtained OCR values were

normalized to the total protein concentration in each

well, measured using the bicinchoninic acid assay

(Sigma-Aldrich). After the experiment, the medium

from each well was collected in a separate microtube

and centrifuged for 10  min at 12,000g to precipitate

detached cells. Also, 10  μl of 2x RIPA buffer (Milli-

pore, Germany) was added to the bottom of each

well to lyse the attached cells. The resulting lysate

was combined with the pellet obtained by centrifug-

ing the culture medium from the same well, and the

pooled lysate was assessed for protein content. The

results of OCR measurements were analyzed using

the Seahorse XFp software (Wave 2.6.1, Seahorse Bio-

science), according to the recommendations for the

Seahorse XF Cell Mito Stress Test Kit (103015-100,

Agilent, USA). Basal respiration was calculated as the

difference between the OCR values after succinate

addition and after addition of a mixture of rotenone,

antimycin, and 2-DG. Respiration associated with ATP

production was calculated as the difference between

the OCR values after succinate addition and addition

of oligomycin. Proton leak was calculated as the dif-

ference in OCR values after addition of oligomycin

and addition of rotenone, antimycin, and 2-DG, while

respiratory control was assessed as the ratio of OCR

values after addition of FCCP to those after addition

of oligomycin. The contribution of succinate-based

respiration to the total oxygen consumption was as-

sessed as the ratio of OCR after succinate addition to

the basal OCR (before the addition of rotenone).

Statistical analysis of data was performed us-

ing the GraphPad Prism9 software (version9.5.1). All

values are presented as mean ± standard deviation

(SD). The data were tested for the normal distribu-

tion using the Shapiro–Wilk test. For experiments in-

cluding two experimental groups, the nonparametric

Mann–Whitney U  test or Student’s t-test for normally

distributed data were used. For the experiments in-

cluding four experimental groups, parametric vari-

ables were analyzed using the one-way analysis of

variance (ANOVA) with the Tukey’s multiple compar-

ison test; nonparametric variables were analyzed us-

ing the Kruskal–Wallis test with the Dunn’s multiple

comparison test.

RESULTS

Succinate promotes the viability of renal epi-

thelial cells and astrocytes under both physiologi-

cal and serum deprivation conditions. We evaluat-

ed the effect of a 48  h incubation with succinate on

the viability of renal epithelial cells and astrocytes

under both physiological and serum deprivation

conditions. Under physiological conditions, succinate

BUYAN et al.1988

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 1. Effect of succinate on the viability of renal epithelial cells and astrocytes after 48h of incubation under physiological

(a, c) or serum deprivation (b, d) conditions. Cell viability was evaluated using the MTT assay. Data are presented as optical

density (OD) at 595 nm for renal epithelial cells (a, b) and astrocytes (c, d); dotted line (b, d) shows the viability of intact

cells; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 compared to the control succinate-free group.

significantly increased the viability of renal epithe-

lial cells at all tested concentrations (Fig.  1a). The

maximal effect was observed at the succinate con-

centration of 50 mM, which increased cell viability

more than two-fold compared to the untreated con-

trol. Serum deprivation for 48  h reduced the viability

of renal epithelial cells by 71%. Succinate markedly

enhanced cell viability upon serum deprivation; the

treatment with 50  mM succinate restored cell viabil-

ity to the levels comparable to those observed under

physiological conditions (Fig.  1b). However, 100  mM

succinate exhibited a toxic effect in serum-deprived

renal epithelial cells, whereas under normal condi-

tions, the same concentration produced a positive

effect (although less pronounced compared to other

tested concentrations) (Fig. 1, a, b).

Succinate also improved the viability of astro-

cytes, albeit to a lesser extent than that of renal ep-

ithelial cells. Under physiological conditions, the via-

bility of astrocytes was significantly enhanced at 12.5,

50, and 100  mM succinate (Fig.  1c) (1.25- and 1.5-fold

at 50 and 100  mM, respectively). Serum deprivation

reduced the viability of astrocytes by ~50%, where-

as the treatment with 100  mM succinate completely

abolished this detrimental effect (Fig.  1d). Overall,

succinate exerted a pro-survival effect in both cell

types, but it was more pronounced in renal epithe-

lial cells.

Succinate treatment reduces cell death in re-

nal epithelial cells, but not in astrocytes. The effect

of succinate on cell death in renal tubular epithelial

cells was further assessed using propidium iodide (PI)

and Calcein AM staining (Fig.  2). Serum deprivation

markedly increased the proportion of PI-positive cells

(Fig.  2, b, d-f), whereas 48-h incubation with succi-

nate significantly reduced the percentage of dead

cells (Fig. 2, b, e). In contrast, succinate failed to at-

tenuate the death of serum-starved astrocytes (Fig.  2,

d, f). These results supported the data of MTT as-

say, indicating that reduced viability during serum

deprivation was due to cell death, predominantly via

necrosis, as well as the ability of succinate to sub-

stantially improved the survival of renal tubular epi-

thelial cells under damaging conditions.

Succinate increases mitochondrial transmem-

brane potential in renal epithelial cells under

both physiological and serum deprivation condi-

tions. Mitochondrial transmembrane potential was

measured to evaluate the effect of succinate on the

status of mitochondria (Fig.  3). A significant increase

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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

Fig. 2. The effect of succinate on the death of renal epithelial cells and astrocytes under physiological and serum depri-

vation conditions. Renal epithelial cells (a, b) and astrocytes (c, d) were stained with Calcein AM and PI after 48 hours of

incubation with succinate under physiological (a, c) and serum deprivation (b, d) conditions. Quantification of PI-positive

renal epithelial cells(e) and astrocytes(f) following succinate treatment; ****p<0.0001 compared to 0mM succinate within

the same experimental group; # p < 0.05 compared to the group exposed to physiological conditions.

BUYAN et al.1990

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Fig. 3. Changes in the mitochondrial transmembrane potential of renal epithelial cells and astrocytes after succinate treat-

ment under physiological and serum deprivation conditions. Mean TMRE fluorescence in renal epithelial cells (a) and

astrocytes (b). Representative confocal images of TMRE-stained renal epithelial cells (c) and astrocytes (d). ** p < 0.01;

**** p < 0.0001 vs. 0 mM succinate under physiological conditions; # p < 0.05 vs. corresponding groups under normal

conditions.

in the transmembrane potential was observed in re-

nal epithelial cells (Fig.  3, a, c), but not in astrocytes

(Fig.  3, b, d), following succinate treatment under

control conditions. Serum deprivation for 48  h led to

a substantial decrease in the TMRE fluorescence in

both renal epithelial cells and astrocytes (Fig.  3). The

treatment with 50  mM succinate under serum depri-

vation conditions improved mitochondrial potential

in renal epithelial cells (Fig.  3, a, c), but failed to ex-

ert a similar effect in astrocytes (Fig.  3, b, d).

Respiratory response of kidney epithelial cells

and astrocytes to succinate treatment. The effects

of exogenous succinate on the respiration parameters

were studied to identify possible mechanisms under-

lying the differences in the cytoprotective effect of

succinate in kidney epithelial cells and astrocytes

(Fig.  4). To measure the OCR, the culture medium

was replaced with a reaction medium containing

no substrates for the respiration or glycolysis, after

which cells were analyzed using a Seahorse XF an-

alyzer after sequential addition of respiratory sub-

strates and inhibitors (see Materials and Methods).

The basal respiration rate was significantly higher in

astrocytes than in kidney epithelial cells (Fig.  4, a, b).

At the same time, renal epithelial cells showed lower

proton leak (Fig.  4d) and, therefore, higher respira-

tory control compared to astrocytes (Fig.  4e). These

findings suggest that the higher basal respiratory rate

of astrocytes may be due to a greater uncoupling of

respiration and oxidative phosphorylation. Never-

theless, both cell types utilized exogenous succinate

as a respiration substrate, as demonstrated by the

OCR increase following succinate addition after rote-

none-mediated inhibition of complexI (Fig.4f). Nota-

bly, succinate utilization was faster in epithelial cells,

as indicated by the kinetics of OCR response (Fig.  4f),

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Fig. 4. The effect of exogenous disodium succinate on the respiration of astrocytes and renal epithelial cells. a)OCR curves

following addition of disodium succinate to the cells in the absence of complex I inhibition. b-e) Quantitative analysis of

mitochondrial respiration parameters calculated from the OCR changes after addition of respiration inhibitors and uncou-

pler: basal respiration(b), ATP-linked respiration(c), proton leak(d), and respiratory control(e). f)OCR curves showing the

response to disodium succinate following rotenone-induced complex I inhibition. g)The contribution of succinate-dependent

respiration to the total oxygen consumption in astrocytes and renal epithelial cells; *p < 0.05; *** p < 0.001.

BUYAN et al.1992

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and its contribution to the oxygen consumption was

more significant than in astrocytes (Fig. 4g).

DISCUSSION

The brain and kidneys are among the organs

with the highest basal energy metabolism. Numer-

ous studies have shown that the optimal function-

ing of brain cells depends directly on the activity

of mitochondria, while mitochondrial dysfunction

contributes to or exacerbates brain pathologies [14].

Although neurons are very metabolically active cells,

astrocytes demonstrate a high glycolytic activity in

order to supply neurons with metabolic substrates

[15]. Nevertheless, mitochondria are considered an

important factor in the regulation of astrocyte func-

tions. It has been shown that during ischemic injury,

changes in the mitochondrial function of astrocytes

are closely associated with impaired brain homeosta-

sis, defects in glutamate and fatty acid metabolism,

regulation, formation of reactive oxygen spe-

cies (ROS), and induction of inflammation [16]. The

kidneys are also among the most energy-consuming

organs, especially renal tubular cells [17, 18]. These

cells are particularly vulnerable to the mitochondrial

dysfunction, which can impair kidney function [19].

Despite the fact that bioenergetic characteristics

of various cell types can differ, mitochondrial dys-

function is a central component of many, if not all,

pathological conditions associated with a deficiency

of nutrient substrates and oxygen. An insufficient

supply of glycolytic and respiratory substrates dis-

rupts oxidative phosphorylation [20], leading to the

destabilization of electron transport chain complexes,

electron leakage, and excessive ROS generation [21].

This creates a vicious cycle, as oxidative stress po-

tentiates further mitochondrial damage, thus increas-

ing ROS production, critically reducing ATP synthesis

[22, 23], and ultimately activating intrinsic apoptotic

pathway, which results in cell death and, finally, or-

gan dysfunction. Therefore, strategies aimed at main-

taining energy homeostasis, such as replenishing me-

tabolite deficiencies with exogenous substrates, are

considered as a promising approach for correcting

mitochondrial dysfunction and preventing cell death.

In this study, serum (FBS) deprivation was used to in-

duce the damage and death of kidney and brain cells

as an in vitro model of cell damage and to examine

the possibility of metabolic correction of the observed

pathological changes with succinate. Low concentra-

tions or absence of FBS in the medium lead to the

activation of signaling molecules of the intrinsic mito-

chondrial apoptotic pathway that involves proteins of

the Bcl-2 family [11]. The absence of FBS also causes

ROS generation, DNA damage, and activation of apop-

totic processes [2]. After serum deprivation, cells con-

sume nutrients in particular, metabolites of the TCA

cycle (e.g., succinate) at a higher rate [24]. In the

acute phase of injury, maintaining a normal amount

of energy substrates is very important, as ROS can

impair the functioning of succinate dehydrogenase

and other respiratory chain complexes, which may

disrupt normal mitochondrial function even after the

nutrient supply restoration [25].

In this study, we investigated the effects of di-

sodium succinate on brain astrocytes and renal ep-

ithelial cells. Disodium succinate was used to avoid

acidification of the culture medium. Although succi-

nate is widely used in clinical practice, its organ-spe-

cific effects, optimal dosage, and effective therapeutic

regimens for various pathologies have not been fully

explored.

Mammalian cells take up exogenous succinate

primarily via sodium-dependent dicarboxylate trans-

porters (NaDCs) of the SLC13A family [26]. Two main

classes of NaDCs have been identified, that differ in

their affinity for succinate: low-affinity transporters

with the Michaelis constant (K

) for succinate of

~0.5  mM, and high-affinity transporters with a K

of ~25  μM [27]. These transporters are expressed in

various tissues, including astrocytes, neurons, kidney

cells, hepatocytes, and immune cells, and facilitate

the entry of succinate and other dicarboxylates into

cells, where these compounds are incorporated into

energy metabolism [28].

Disodium succinate increased the viability of re-

nal epithelial cells and astrocytes under both stan-

dard conditions and serum deprivation-induced stress.

However, the beneficial effect of succinate was more

pronounced in renal epithelial cells (Fig.  1). These

data are consistent with the previously obtained re-

sults showing that succinate improved mitochondrial

function in glial cells [29] and reduced neuroinflam-

mation in experimental intracerebral hemorrhage

[30]. One of the key biochemical markers of brain

injury is the lactate/pyruvate ratio in brain tissue.

An increase in this ratio reflects a shift towards an-

aerobic glycolysis and oxidative stress development.

Clinical administration of succinate-containing formu-

lations to patients with traumatic brain injury (TBI)

decreased the lactate/pyruvate ratio, indicating im-

proved redox balance and increased glucose utiliza-

tion efficiency [31-33]. This effect is thought to result

from the stimulation of the TCA cycle and oxidative

phosphorylation, leading to the increased pyruvate

levels and decreased pyruvate reduction to lactate

[33]. Furthermore, exogenous succinate improved

glutamatergic synaptic transmission in rat hippocam-

pal vital slices, which may be due to the improved

energy supply to presynaptic terminals [34]. In con-

trast to the nervous system, the effects of succinate

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BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

on the renal tissue have been studied much less.

However, succinate can also improve mitochondrial

function by reducing oxidative stress and replenish-

ing deficient energy substrate in the cases of renal

proximal tubule cell damage [35].

We demonstrated that exogenous succinate main-

tained cellular energy status by significantly increas-

ing the mitochondrial transmembrane potential. How-

ever, this effect was specific to renal epithelial cells

(Fig.  3). To understand why succinate had a more

pronounced positive effect on renal epithelial cells vs.

astrocytes, we compared the respiration parameters

under succinate energization (Fig. 4). The basal bio-

energetic characteristics of astrocytes and renal epi-

thelial cells differed significantly. Astrocytes exhibited

higher basal respiration rates, likely due to a higher

degree of mitochondrial uncoupling rather than to

a more active oxidative phosphorylation. Oligomy-

cin-sensitive respiration was similar between the two

cell types, whereas the proton leak and respiratory

control values (Fig.  4, d, e) indicated the occurrence

of the futile cycle in astrocytes. This might reflect the

reliance of astrocytes on glycolysis to fuel the astro-

cyte–neuron lactate shuttle, with an unchanged rate

of oxidative phosphorylation. Since the cells under the

experimental conditions had only succinate as a respi-

ratory substrate, it can be assumed that it was more

efficiently utilized by renal tubular cells. Oxygen con-

sumption values in the case of complex I inhibition

by rotenone following addition of succinate, showed

that succinate significantly increased the respiration

rate (Fig.  4f). This confirms the functional involve-

ment of exogenous succinate in the electron transport

chain. Moreover, the increase in the oxygen consump-

tion in tubular epithelial cells after succinate addition

occurred more rapidly than in astrocytes (Fig.  4f),

and the overall contribution of succinate-dependent

respiration in renal epithelial cells was significantly

higher than in astrocytes (Fig.  4g). The cell type-spe-

cific efficacy of disodium succinate can be explained

by both differences in the expression of dicarboxyl-

ate transport systems and higher activity of succinate

dehydrogenase in renal mitochondria. These features

make the energy metabolism of tubular epithelial

cells dependent on the availability of succinate [36].

In addition to its direct role in energy metab-

olism, the protective effect of succinate under the

damaging conditions may be mediated by several

other mechanisms, such as activation of the SUCNR1

receptor (GPR91) and inhibition of prolyl hydroxylas-

es (PHDs). The SUCNR1 receptor is widely expressed

in brain cells, in particular, astrocytes, microglia, and

neurons, as well as in renal tubular epithelial cells

[33, 37]. SUCNR1 is thought to be involved in tissue

adaptation to adverse conditions, including stimula-

tion of cell proliferation, migration, and angiogenesis

[38,  39]. Notably, the interaction of extracellular suc-

cinate with SUCNR1 can induce expression of SLC13A

family genes, which ensures succinate transport into

the cells [40]. This creates a positive feedback loop,

where extracellular succinate stimulates its own up-

take, enhancing both its metabolic and signaling ef-

fects. Moreover, after entering the cytosol, succinate

can inhibit 2-oxoglutarate-dependent dioxygenases,

primarily PHDs [41,  42], which are responsible for

the oxygen-dependent degradation of hypoxia-in-

ducible factor 1-alpha (HIF-1α). Inhibition of PHDs

stabilizes HIF-1α even under normoxic conditions,

enabling its nuclear translocation [43] and activa-

tion of genes involved in the adaptation to hypox-

ia and regulation of mitochondrial function [44].

In particular, this triggers the synthesis of cytochrome

oxidase subunit  2 (COX4-2), which replaces the con-

stitutive form COX4-1, thus increasing the efficien-

cy of complex IV at low oxygen concentrations [45].

It is worth noting that, in addition to succinate, oth-

er exogenous metabolites and their derivatives with

a higher bioavailability are actively studied. One ex-

ample is diacetoxymethyl succinate (NV118), which

shows a pronounced cytoprotective effect due to the

improvements in the mitochondrial metabolism in

the models of damage induced by carbon monox-

ide, cyanides, amiodarone, N-methylcarbamate, and

metformin [46-52]. Other alternative substrates (e.g.,

sodium fumarate) reduce cell death during hypoxia,

mostly by activating alternative pathways rather than

restoring mitochondrial ATP synthesis [53]. Dimeth-

yl fumarate has demonstrated the renoprotective ef-

fect against cyclosporine A- and lipopolysaccharide

(LPS)-induced toxicity, mediated by suppression of ox-

idative stress and inflammation, as well as enhance-

ment of the antioxidant defense [54, 55]. Dimethyl

fumarate also inhibited pyroptosis via modulation

of endoplasmic reticulum stress and the JAK2–STAT3

pathway in a model of contrast-induced nephropathy

[56] and improved mitochondrial function in the case

of di(2-ethylhexyl)phthalate toxicity [57]. The effects

of dimethyl fumarate are most likely explained by the

activation of the NRF2 pathway [58], which accounts

for its neuroprotective potential [59,  60], including

attenuation of neuroinflammation and neuronal cell

death after traumatic brain injury [61,  62]. Similarly,

monomethyl fumarate acts through NRF2, as it sup-

presses cell death and oxidative stress, and reduces

brain edema in a model of ischemic stroke [63-65].

Taken together, these data suggest that TCA cycle me-

tabolites and their derivatives have a considerable

therapeutic potential for ameliorating mitochondrial

dysfunction across a broad spectrum of pathologies

that require multiorgan protection. However, despite

their enhanced membrane permeability, ester deriva-

tives of dicarboxylic acids, including succinate, have

BUYAN et al.1994

BIOCHEMISTRY (Moscow) Vol. 90 No. 12 2025

limitations as therapeutic agents. In particular, com-

monly used alkyl esters, such as dimethyl succinate,

are hydrolyzed by cellular esterases with the release

of corresponding alcohols (e.g., methanol), which may

exert toxic effects. This highlights the critical need

for the development of alternative, safe formulations

capable of enhancing succinate bioavailability with-

out compromising the safety.

CONCLUSION

This study demonstrated a prominent cytopro-

tective potential of exogenous disodium succinate in

cells derived from two highly metabolically active or-

gans – the brain and kidneys – especially after injury.

Succinate significantly increased the viability of both

astrocytes and renal epithelial cells under both phys-

iological and stress conditions. However, its protec-

tive effect was more pronounced in kidney epithelial

cells than in astrocytes. This difference likely reflects

a more pronounced ability of succinate to modulate

the mitochondrial transmembrane potential in kidney

epithelial cells and to support oxidative phosphoryla-

tion when other metabolic substrates are limited, sug-

gesting more efficient utilization of succinate in renal

epithelial cells. These findings support the hypothesis

that succinate, a key TCA cycle intermediate, can al-

leviate cellular energy deficit and prevent cell death.

However, its effects appear to be cell type-dependent,

necessitating careful selection of target pathologies.

Therefore, succinate represents a particularly prom-

ising candidate for the nephroprotective strategies

in acute kidney injuries associated with bioenergetic

crisis.

Abbreviations

2-DG 2-deoxyglucose

ATP adenosine triphosphate

FBS fetal bovine serum

PHD prolyl hydroxylases

PI propidium iodide

ROS reactive oxygen species

TMRE tetramethylrhodamine ethyl ester

Contributions

N. V. Andrianova and E. Y. Plotnikov developed

the concept and supervised the study; M. I. Buyan,

K. S. Cherkesova, N. V. Andrianova, A. A. Brezgunova,

and I. B. Pevzner conducted the experiments; E. Y. Plot-

nikov, N. V. Andrianova, M. I. Buyan, K. S. Cherkeso-

va, A. A. Brezgunova, and I. B. Pevzner discussed

the study results; M. I. Buyan, K. S. Cherkesova, and

N. V. Andrianova wrote the original draft; E. Y. Plot-

nikov, A. A. Brezgunova, and I. B. Pevzner edited

themanuscript.

Funding

This work was supported by the Russian Science Foun-

dation (project no.24-75-10013).

Acknowledgments

The study was conducted on a Zeiss LSM900 confocal

microscope with support from the Moscow State Uni-

versity Development Program.

Ethics approval and consent to participate

Animal study protocols were reviewed and approved

by the Ethics Committee of the Belozersky Institute of

Physico-Chemical Biology, Lomonosov Moscow State

University (protocol no. 006-1/2/2024). All animal

manipulations were performed in accordance with

ARRIVE guidelines.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

Open access

This article is licensed under a Creative Commons At-

tribution 4.0 International License, which permits use,

sharing, adaptation, distribution, and reproduction

in any medium or format, as long as you give appro-

priate credit to the original author(s) and the source,

provide a link to the Creative Commons license, and

indicate if changes were made. The images or other

third party material in this article are included in the

article’s Creative Commons license, unless indicated

otherwise in a credit line to the material. If materi-

al is not included in the article’s Creative Commons

license and your intended use is not permitted by

statutory regulation or exceeds the permitted use,

you will need to obtain permission directly from the

http://creativecommons.org/licenses/by/4.0/.

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