Nutrient substrate deficiency and impaired energy metabolism are hallmarks of a wide range of severe pathologies, including cardiovascular diseases, sepsis, and hemorrhagic shock [1,2]. Impaired tissue perfusion often results in ischemia/reperfusion (I/R) injury, characterized by a drastic reduction in oxygen and energy metabolic substrate delivery due to compromised blood flow [3]. The lack of key energy substrates can be as detrimental as hypoxia itself. Substrate deficiency induces an energetic crisis, disrupts metabolic and biosynthetic processes, and is commonly associated with mitochondrial dysfunction and oxidative stress due to electron transport chain (ETC) impairments. These changes lead to violations in ATP synthesis and failure of ATP-dependent processes and cell homeostasis. Ultimately, metabolic collapse triggers the intrinsic apoptotic pathway or, in cases of severe depletion, leads to necrotic cell death [4].

The kidney, liver, and brain are highly sensitive to disturbances in energy metabolism due to their high and sustained energetic demands. In the brain, astrocytes support the high energy requirements of neurons by supplying lactate as an alternative fuel source via the astrocyte-neuron lactate shuttle [5]. Similarly, renal tubular cells require significant amounts of ATP for the reabsorption of amino acids, glucose, and electrolytes, mediated by ATP-dependent transporters [6]. The liver exhibits high energy expenditure due to its central role in carbohydrate, lipid, and protein metabolism [7]. This metabolic demand is further amplified by the liver’s robust regenerative capacity following injury, which requires sustained metabolic activity [8].

One promising approach to correcting energy imbalance involves the use of exogenous intermediates of the tricarboxylic acid (TCA) cycle. Reducing equivalents, produced by the TCA cycle, are essential for ETC function and ATP replenishment. To preserve mitochondrial integrity and function, a continuous flux of reducing equivalents from the TCA cycle to the ETC must be maintained [9]. Among TCA cycle intermediates, fumarate has attracted attention as a metabolite with cytoprotective potential for treating conditions associated with energy substrate deficiency. Under physiological conditions, fumarate, as a product of succinate oxidation and a substrate of fumarate hydratase, serves as an intermediate of the TCA cycle, thereby supporting cellular energy production [10]. Under pathological conditions, the integrity of the cycle is disrupted, and fumarate, losing its role as a mere metabolic intermediate, instead functions as a signaling molecule. Through the activation of Nrf2, it induces the expression of cytoprotective genes, ultimately attenuating oxidative stress and inflammation [11,12,13]. However, despite increasing interest in fumarate and its derivatives, effective protocols for their use in ischemic pathologies remain insufficiently defined. In particular, the tissue- and cell type-specific effects of fumarate under conditions of metabolic substrate deprivation are poorly understood.

In this study, we aimed to identify organ-specific features of the metabolic responses to ischemic injury and to assess whether exogenous fumarate modulates the viability and mitochondrial function of cells from different organs. To address this, we performed a comparative analysis of transcriptomic data for the brain, kidney, and liver under normal and ischemic conditions, focusing on genes encoding key components of energy metabolism, including proteins of the TCA cycle and ETC. Then, the cellular responses to exogenous fumarate were compared using astrocytes, renal tubular epithelial cells, and hepatic stellate cells under normal and stress conditions in vitro. The effects of various fumarate concentrations on cell viability and mitochondrial transmembrane potential were assessed to identify tissue-specific patterns of metabolic sensitivity and adaptation.

The deficiency of energy substrates is a central pathophysiological mechanism underlying many acute and chronic ischemic conditions. This deficit arises primarily as a consequence of impaired tissue perfusion, which limits the delivery of oxygen and metabolic substrates. One of the most common causes of such substrate deprivation is I/R injury [23]. I/R injury may develop locally due to vascular or circulatory impairments, as in myocardial infarction, stroke, or kidney and liver ischemia. It can also occur at a systemic level during shock caused by massive hemorrhage or severe trauma [24]. In these conditions, sudden intravascular volume loss triggers reduced tissue perfusion, impaired oxygen and nutrient delivery, and ultimately, the development of multiple organ failure [25].

Impaired blood supply is often associated with alterations in mitochondrial homeostasis. A deficit of metabolic substrates and oxygen suppresses mitochondrial function and may be accompanied by deleterious changes in oxidative phosphorylation, mitochondrial membrane potential, and ATP generation. As a consequence, ATP-dependent processes, like ion transport and protein synthesis, are disrupted [26]. Furthermore, the dysfunction of the respiratory chain in ischemia primes the mitochondria for excessive reactive oxygen species generation upon reperfusion, which further exacerbates tissue damage [27,28]. These alterations ultimately lead to cell death, often through necrosis rather than apoptosis, resulting in severe tissue damage. Although energetic stress represents a common challenge across tissues, the cellular responses to energy substrate deficiency are not uniform but are determined by tissue-specific metabolic features and adaptive mechanisms.

Astrocytes play a key role in neurometabolic coupling, supporting neurons by producing lactate and protecting them from oxidative stress [29,30]. Under ischemic conditions, energy deficit triggers a metabolic shift that preferentially affects astrocytes [30,31]. Due to their direct access to glucose from blood vessels, astrocytes increase glucose uptake and activate glycolysis, whereas these processes are less pronounced in neurons [32]. Furthermore, astrocytes demonstrate greater resilience, preserving mitochondrial function during ischemia [33]. Such compensatory mechanisms in the injured brain tissue, in particular after I/R, are consistent with a relative preservation of TCA and ETC expression profiles after I/R (Fig. 2a) [34,35].

Liver cells can flexibly utilize various metabolic substrates, such as fatty acids, glucose, amino acids, or ketone bodies, to fulfill their energy needs [7]. However, rapid replenishment of the ATP pool in liver cells relies on a stable ETC and healthy mitochondrial functions [36]. However, the observed transcriptional downregulation of genes encoding TCA cycle and ETC enzymes (Fig. 4) does not exclude the capacity of hepatocytes to partially compensate for energetic stress through alternative substrate utilization, a feature reflecting the hepatic ability to flexibly switch between energy substrates to compensate for the metabolic deficits. Such metabolic remodeling has consequences not only for hepatocyte energy balance but also for repairing tissue responses after injury. In the liver, these responses involve hepatic stellate cells playing a central role in remodeling processes and maintenance of hepatocyte function. These cells, at the same time, remain sensitive to metabolic and ischemic insults [37]. However, it is important to acknowledge that the use of the LX-2 cell line in isolation represents a limitation of this study. While liver stellate cells possess broad metabolic capabilities and contribute to hepatic metabolic plasticity, they do not fully replicate the metabolic landscape of the whole liver, where hepatocytes predominate.

In contrast to the liver, where metabolic flexibility may partially compensate for energetic stress, renal epithelial cells depend heavily on sustained mitochondrial oxidative metabolism to support energy-intensive functions essential for kidney homeostasis, including the reabsorption of glucose, electrolytes, and water, as well as the precise regulation of acid-base balance [38]. These processes demand substantial ATP production, making mitochondrial energetics critical for renal function. Under glucose deficiency, renal epithelial cells can shift toward fatty acid oxidation to replenish the pool of acetyl-CoA, because the oxidative phosphorylation remains central to their energy metabolism [39]. Consistent with this metabolic dependency, our transcriptome analysis reveals significant suppression of the gene expression of both mitochondrial respiratory chain complexes and TCA cycle enzymes in kidney tissue after I/R (Fig. 3). Compared to the liver and brain, this transcriptional response was more pronounced, indicating a high vulnerability of the kidney to ischemic damage. Consistent with this interpretation, our analysis revealed increased Complex IV protein levels in astrocytes under deprivation conditions, while kidney epithelial cells showed increased GAPDH levels (Supplementary Fig. S4). Although these observations are limited to a small set of markers, they support the concept of organ-specific bioenergetic adaptation suggested by the transcriptomic data.

The deficiency of TCA cycle metabolites during ischemia poses a key challenge for the restoration of mitochondrial respiration, cellular metabolism, and normal organ function [40]. Our data underscore that metabolic flexibility, i.e., the ability to maintain energy homeostasis by remodeling major bioenergetic pathways, is not a universal property but rather a tissue-specific feature. Following injury, the inherent metabolic specialization of organs undergoes significant reorganization, which is reflected in substantial shifts in gene expression profiles (Fig. 1). This observation aligns with a number of previous studies describing injury-associated metabolic reprogramming in both acute injuries [41,42] and chronic pathologies [43,44,45]. Thus, while nervous tissue maintains or even upregulates the expression of TCA cycle and ETC genes in response to ischemia, kidney and liver tissues exhibit varying degrees of transcriptional repression of these pathways. Such transcriptomic patterns likely reflect fundamental differences in the organs’ metabolic architecture rather than uniform sensitivity to energy substrate deficiency. This, in turn, may contribute to the differential vulnerability of various tissues to the availability of TCA cycle intermediates, such as fumarate.

The protective effect of fumarate-related compounds has been reported for a number of pathologies. Administration of sodium fumarate in the deoxycorticosterone acetate-salt hypertension model reduced the expression of the damage marker kidney injury molecule-1 (KIM-1) and restored kidney NO production [46]. In cellular models of hypoxia, it decreased cell death and partially restored the activity of ETC complex 1 [47]. In parallel, administration of bioavailable fumarate derivatives, particularly dimethyl fumarate and monomethyl fumarate, demonstrated cytoprotective effects in various pathologies. These effects have been largely attributed to activation of Nrf2-dependent antioxidant signaling pathways, resulting in reduced oxidative stress and improved cellular survival in models of kidney [48], hepatic [49], and brain injury [50,51,52]. Beyond mitigating oxidative stress, dimethyl fumarate has been reported to improve mitochondrial function in acute kidney injury, specifically by restoring succinate dehydrogenase activity [53]. There is growing evidence that fumarate itself can directly modulate the Nrf2 transcriptional pathway. For instance, sodium fumarate supplementation significantly restored the expression of Nrf2-associated antioxidant genes, including classical targets such as Hmox1 and Nqo1, which had been suppressed in the model of metabolic stress [54]. The findings are supported by results from transgenic animals, in particular mice with knockout of fumarate hydratase, in which activation of Nrf2 signaling was also detected in the heart and kidneys [12,55].

In contrast to dimethyl fumarate and monomethyl fumarate, whose protective effects are largely attributed to well-characterized signaling pathways, the cellular actions of fumarate itself remain less clearly defined. Our data indicate that the organ-specific effects of fumarate are largely associated with mitochondrial function (Fig. 8, Fig. 9 and Fig. 10). A limitation of this study is that we assessed only changes in mitochondrial membrane potential across different cell types in response to fumarate, without evaluating the corresponding changes in mitochondrial ultrastructural morphology. Nevertheless, fumarate accumulation can exacerbate organ damage [56,57]. Consistent with this, our data show that the highest concentration of fumarate (100 mM) induced cytotoxicity, most notably in kidney and liver cells under both normal and serum-deprived conditions (Fig. 7b,c,e,f). The use of disodium fumarate had a limitation inherent in the experimental design of our in vitro model regarding the physiological relevance of the millimolar fumarate concentrations employed. Fumarate derivatives are established therapeutic agents for hypoxic and critical conditions, psoriasis and multiple sclerosis and are applied at lower concentrations due to their better permeability [58,59,60]. However, in this study it was important to exclude the hydrolysis products of methyl fumarate derivatives [61], so the disodium salt of fumaric acid was used.

The combined analysis of DAPI (Fig. 6 and Fig. 7) and MTT (Fig. S5) data revealed a tissue-specific dynamic of the response to fumarate. In astrocytes, both methods showed a consistent maximum protective effect under deprivation at 50 mM fumarate (Fig. 7a,d and Fig. S5a,d). In kidney epithelial cells, the MTT test revealed a significant increase in viability under deprivation at 50 mM fumarate (Fig. S5e), while no significant increase in nuclear counting was revealed (Fig. 7e). This may indicate a primary influence on metabolic activity rather than on the proliferation and restoration of kidney cells. In hepatic stellate cells, the data from both DAPI-staining and MTT methods correlate, demonstrating a shift in the optimum protective effect to a lower concentration range of 12.5–25 mM under both normal and stress conditions (Fig. 7c,f and Fig. S5c,f). Based on these findings, we propose that the tissue-specific action of fumarate may be attributed to its bioavailability, transport, or intracellular handling.

In health and disease, fumarate bioavailability is largely determined by the number of transporters and receptors with different affinities for this metabolite, as well as the activation of signaling cascades specific to each cell type. Pharmacodynamic analyses have demonstrated non-uniform tissue distribution of fumarate derivatives [62], suggesting the hypothesis of tissue-specific distribution of fumarate receptors, including hydroxyl carboxylic acid receptor-2 (HCAR2, also known as GPR109a). HCAR2 expression is significantly increased under inflammatory and hemolytic conditions, indicating its role in metabolic adaptation under stress [63]. In addition to its established role in lipid metabolism, HCAR2 participates in inflammatory response [64] through AMPK/SIRT1 signaling, which regulates microglia switching toward an anti-inflammatory and neuroprotective phenotype [65]. While direct involvement of this pathway was not examined in the present study, it is conceivable that high extracellular fumarate concentrations under deprivation conditions may engage similar signaling mechanisms in astrocytes, potentially contributing to the observed changes in cell number (Fig. 7d). Consistent with these results, the neuroprotective effect of fumarate and its derivatives has been demonstrated recently in cortical neuron cultures under oxygen-glucose deprivation [66] and in stroke models in vivo [51].

Beyond its metabolic role, fumarate can influence the epigenetic profile of cells by regulating the activity of Ten-Eleven Translocation DNA demethylases (TET) and Jumonji C domain-containing histone demethylases (JmjC). By competing with α-ketoglutarate, elevated fumarate levels inhibit many α-ketoglutarate-dependent demethylases, leading to changes in gene expression through changes in DNA and histone methylation patterns [67]. In our study, exposure to fumarate at concentrations ≥12.5 mM under deprivation conditions resulted in a decrease in the number of renal epithelial cells (Fig. 7e), which may be associated with the effect of fumarate on the epigenetic landscape of cells. It has been shown that under chronic metabolic stress, excess fumarate can impair the function of demethylases, promoting long-term changes in gene expression that affect inflammation and tissue repair [68].

Another possible mechanism regulating the tissue-specific action of fumarate involves fumarate hydratase 1 (FH1), which controls the accumulation of this metabolite in the cell. Genetic deficiency of FH1 leads to pathological fumarate accumulation and profound alterations in cellular signaling and epigenetic regulation, which result in the development of hereditary leiomyomatosis and renal cell carcinoma (HLRCC) [69]. While long-term deprivation does not affect FH1 expression, it significantly increases the expression of an alternative FH variant, which is associated with the cellular response to stress [70]. Thus, under deprivation conditions, fumarate can suppress renal epithelial proliferation by inactivating several demethylases and by inducing the FH variant, associated with cellular stress response. This may partially explain the observed decrease in the number of renal epithelial cells after incubation with fumarate in stress conditions (Fig. 7e).

In addition to intracellular metabolism, an important role in the cell exposure to fumarate plays in the regulation of its transport. Fumarate is transported by the members of the solute carrier family (SLC), which are involved in transporting various TCA cycle intermediates. One of these transporters is SLC13A3, which is important for the transport of fumarate and other dicarboxylates in astrocytes, renal epithelial cells, and likely hepatic stellate cells [71,72,73]. Its dysfunction is associated with neurological and metabolic disorders, as well as the pathogenesis of liver diseases, making it a promising target for further research and therapy [71,72,73].

Replenishing TCA cycle intermediates, including fumarate, has been proposed as a promising strategy to support cellular metabolism during ischemic injury. Our data showed a tissue-specific effect of fumarate in vitro under both normal and pathological conditions. Under serum deprivation conditions, fumarate influenced mitochondrial status, as indicated by an increase in potential-dependent TMRE accumulation in astrocytes, renal epithelial cells, and hepatic stellate cells. However, a similar effect on mitochondria was accompanied by tissue-specific changes in cell number. These cell-specific, multidirectional effects of fumarate likely reflect distinct regulatory mechanisms, including differences in signaling pathways, epigenetic regulation, intracellular metabolism, and transport. Therefore, developing effective metabolite-based therapies for ischemic injury requires not only targeting energy imbalance but also accounting for the metabolic specialization and functional state of target cells.