Protective effects of adenosine triphosphate, thiamin, thiamin pyrophosphate, and their combination against linezolid-induced renal injury and lactic acidosis in rats

Abstract

Linezolid, an oxazolidinone antibiotic effective against Gram-positive pathogens, may cause nephrotoxicity and lactic acidosis during prolonged therapy. This experimental study investigated the protective effects of adenosine triphosphate (ATP), thiamin, thiamin pyrophosphate (TPP), and their combination (ATTP) on linezolid-induced renal injury and lactic acidosis in rats. Thirty-six male Wistar rats were randomly divided into six groups (n = 6): healthy control (HG), linezolid only (LZD), ATP+linezolid (ATLZD), thiamin + linezolid (TLZD), TPP+linezolid (TPLZD), and ATP+thiamin + TPP+linezolid (ATTPL). Linezolid (125 mg/kg, orally) was administered twice daily, while ATP (4 mg/kg), thiamin (20 mg/kg), and TPP (20 mg/kg) were given intraperitoneally once daily for 28 days. At the end of treatment, kidney tissues were examined for oxidative stress markers [malondialdehyde (MDA), total glutathione (tGSH), superoxide dismutase (SOD), catalase (CAT)] and histopathology, and blood samples were analyzed for blood urea nitrogen (BUN), creatinine, and lactate. Linezolid increased oxidative stress, suppressed antioxidants, and elevated BUN, creatinine, and lactate levels. ATP partially improved the oxidative balance in renal tissue but failed to prevent hyperlactatemia and impaired renal function. Thiamin did not produce significant changes. TPP markedly improved oxidative stress markers and reduced renal dysfunction. The triple combination provided the most pronounced protection, restoring antioxidant defenses, kidney function, and lactate levels to near-control values. Histopathological evaluation revealed marked tubular degeneration, interstitial hemorrhage, and mononuclear cell infiltration in the linezolid group, which were markedly improved by TPP and combination therapy. These findings indicate that TPP protects against linezolid-induced nephrotoxicity and lactic acidosis, with its efficacy further enhanced by ATP.

1. Introduction

Linezolid is a synthetic antibiotic belonging to the oxazolidinone class, effective against Gram-positive microorganisms [1]. It has been approved for the treatment of hospital-acquired and community-acquired pneumonia, skin and soft tissue infections, and infections caused by vancomycin-resistant Enterococcus faecium [2]. Linezolid exerts its effect by inhibiting bacterial protein synthesis. This inhibition occurs through binding to a specific site on the 50S subunit of the bacterial 23S ribosomal RNA, thereby preventing the formation of the functional 70S initiation complex [1]. Although linezolid is a broad-spectrum antibiotic effective against resistant pathogens, it has been associated with serious adverse effects, including thrombocytopenia, optic neuropathy, peripheral neuropathy, and lactic acidosis [3]. Cases of renal failure associated with linezolid use have also been reported [4]. Savard et al. documented a case of acute interstitial nephritis accompanied by eosinophilia and systemic symptoms during linezolid therapy [5]. Moreover, linezolid has been shown to exert nephrotoxic effect by increasing levels of lactic acid, serum urea, creatinine, MDA, and proinflammatory cytokines, while decreasing CAT and GSH levels [6].

The kidneys play a crucial role in lactate metabolism, and under conditions of hyperlactatemia, lactate elimination largely occurs via this metabolic route. However, in cases of pronounced hyperlactatemia, the renal capacity to eliminate lactate diminishes [7]. If left untreated, lactic acidosis can lead to a range of pathological conditions, including acute kidney injury, pancreatitis, liver failure, and cardiovascular disorders [8]. Despite the information available in the literature, the underlying mechanisms of linezolid toxicity have not been fully elucidated. Recent studies have reported that linezolid may decrease intracellular ATP concentrations [9], suggesting that reduced ATP levels could play a key role in the manifestation of its toxic effects. It is emphasized that reductions in the production of ATP, which consists of an adenine base, a ribose sugar, and three phosphate groups, may lead to an increase in oxidative stress [10,11].

Thiamin, also referred to as vitamin B1, is being evaluated in our study for its potential protective role against nephrotoxicity potentially associated with linezolid treatment [12]. This water-soluble vitamin is naturally found in red meat, legumes, whole grains, and nuts [13]. Thiamin deficiency has been shown to reduce ATP synthesis and impair oxidative metabolism [14]. Thiamin is converted into its active metabolite, TPP, by the enzyme [15]. TPP serves as a cofactor for the enzyme pyruvate dehydrogenase (PDH) and plays a critical role in the conversion of pyruvate to acetyl-coenzyme A. Therefore, in the case of TPP deficiency, pyruvate is converted to lactate, leading to hyperlactatemia, also known as lactic acidosis [16]. Administration of TPP has been reported to prevent lactate accumulation, improve glucose metabolism, and enhance aerobic capacity [17]. Additionally, TPP has demonstrated antioxidant properties and has been shown to protect tissues against oxidative stress [14]. Current literature suggests that ATP, thiamin, and its active form TPP may have potential therapeutic value in preventing linezolid-induced nephrotoxicity and lactic acidosis; however, to date, the effects of ATP, thiamin, TPP, or ATTP on linezolid-induced renal toxicity and lactic acidosis in rats have not been systematically investigated. Therefore, in this study, the effects of ATP, thiamin, TPP, and ATTP on linezolid-induced kidney injury and lactic acidosis were evaluated biochemically, and histopathological changes in kidney tissues were examined. Within this framework, we tested the following hypotheses: (H1) Linezolid produces a concurrent energy–redox injury phenotype in the kidney (increased MDA, decreased tGSH, SOD, and CAT activities, and elevated BUN, creatinine, and lactate levels); (H2) TPP is superior to thiamin because it bypasses the TPK–dependent activation step and thereby restores PDH flux; (H3) ATP alone primarily improves redox status but has a limited effect on lactic acidosis; (H4) the ATP+thiamin + TPP combination provides additive protection by supporting ATP pools, supplying the active PDH cofactor, and strengthening the antioxidant defense.

2. Materials and methods

2.1. Animals

The experimental research was carried out on 36 male Wistar albino-type rats, aged between 9 and 10 weeks, with body weights ranging from 281 to 290 grams. The animals were sourced from the Experimental Animals Application and Research Center of Erzincan Binali Yıldırım University (Erzincan, Turkey). Six groups were formed by randomly assigning the animals, ensuring that the mean body weight was similar across all groups. Before the initiation of the experiment, the rats were housed in groups of six in standard wire laboratory cages (20 cm in height, 35 cm in width, and 55 cm in length; floor area: 1,925 cm²) to facilitate acclimatization to laboratory conditions. The rats were kept under controlled environmental conditions, including a 12-h light/12-h dark cycle, a constant temperature of 22 °C, and relative humidity between 30% and 70%. Animals were granted free access to tap water and a commercially available pelleted feed (laboratory animal chow; Bayramoglu Stock Company, Erzurum, Turkey) throughout the study. The study was conducted in accordance with the European Directive 2010/63/EU on the protection of animals used for scientific purposes (Approval ID: 2016-24-199) and followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines [18]. Experimental procedures involving animals took place within the laboratories of the Experimental Animal Application and Research Center at Erzincan Binali Yıldırım University. All animal-related procedures received prior approval from the local Ethics Committee for animal experiments at Erzincan Binali Yıldırım University (Erzincan, Turkey; Session Date: 29.05.2025; Session No: 2025/05; Approval No: 26).

2.2. Chemicals

The linezolid (Zyvoxid^® 600 mg tablet) used in this study was obtained from Pfizer Pharmaceuticals (Istanbul, Turkey), thiopental sodium (Pental Sodyum^® 0.5 g vial) from IE Ulagay (Istanbul, Turkey), ATP (10 mg/mL vial) was supplied by Zdorove Narodu (Kharkiv, Ukraine), thiamin (Thiamin chloride^® 50 mg/mL solution for injection) and thiamin pyrophosphate (Cocarboxylase hydrochloride^® 50 mg/2 mL solution for injection) were sourced from BioPharma (Kiev, Ukraine).

2.3. Experimental design

The sample size was calculated to ensure the minimal use of animals, in strict adherence to the 4 R (Reduction, Refinement, Replacement, and Responsibility) principles [19]. Criteria including slumped posture, reduced mobility, and injuries inflicted by other animals were employed to exclude subjects throughout the experiment and corresponding data points during analysis. No exclusions were made during the course of the experiment. A random number table was used to generate the randomization sequence, and both cages and animals were assigned identification numbers to minimize potential confounding variables.

2.4. Experimental groups

Animals were randomly allocated into six experimental groups: healthy control (HG), linezolid alone group (LZD), ATP combined with linezolid group (ATLZD), thiamin combined with linezolid group (TLZD), thiamin pyrophosphate combined with linezolid group (TPLZD), and a combination of ATP, thiamin, thiamin pyrophosphate, and linezolid group (ATTPL).

2.5. Experimental procedure

ATP (4 mg/kg) [20], thiamin (20 mg/kg) [21], TPP (20 mg/kg) [22], and a combination of ATP, ATTP were administered intraperitoneally (IP) to the ATLZD (n = 6), TLZD (n = 6), TPLZD (n = 6), and ATTPL (n = 6) groups, respectively. Saline (0.9% NaCl) was administered as a vehicle to HG (n = 6) and LZD (n = 6) groups via the same route. One hour after administration of ATP, thiamin, TPP, ATTP, or saline, linezolid was administered orally at a dose of 125 mg/kg to the LZD [23,24], ATLZD, TLZD, TPLZD, and ATTPL groups. Linezolid was administered twice daily at 12-h intervals, while ATP, thiamin, TPP, and ATTP were administered once daily for a period of four weeks. At the end of the treatment period, all rats were euthanized under deep anesthesia, and their kidneys were excised. Biochemical analyses were performed on kidney tissues to determine levels of MDA, tGSH, SOD, and CAT. Additionally, kidney tissues were subjected to histopathological analysis to assess structural alterations. Before euthanasia, blood was drawn from the tail veins of the rats for the quantification of BUN, creatinine, and lactate levels.

2.6. Biochemical analyses

2.6.1. Sample preparation

After being rinsed with physiological saline, tissue samples were ground in liquid nitrogen under cryogenic conditions and then homogenized. The resulting supernatants were used to determine levels of MDA, GSH, SOD, CAT, and total protein.

2.6.2. Determination of MDA, GSH, SOD, CAT, and protein levels in renal tissue

The concentrations of MDA, total GSH, and SOD in the tissue homogenates were determined using commercially available rat ELISA kits, following the manufacturer’s instructions. The kits used were MDA (Cat. No.10009055), GSH (Cat. No.703002), and SOD (Cat. No.706002), all obtained from Cayman Chemical Company (Ann Arbor, MI, USA). The determination of CAT activity followed a standardized procedure adapted from the methodology proposed by Goth [25]. Protein quantification was carried out using the Bradford method, which involves spectrophotometric analysis at 595 nm [26].

2.6.3. Measurement of blood urea nitrogen levels as a marker of renal function

Serum urea concentrations were quantitatively measured using a spectrophotometric technique on the Cobas 8000 automated-analyzer (Roche Diagnostics, Mannheim, Germany). BUN values were derived by multiplying the measured urea concentration by a conversion factor of 0.48. Within this enzymatic kinetic assay utilizing urease and glutamate dehydrogenase (GLDH), urea undergoes hydrolysis to yield ammonium ions and carbonate, as follows: Reaction 1: Urea + 2H₂O → (via urease) 2NH₄⁺ + CO₃^2-. During the second enzymatic transformation, GLDH catalyzes the conversion of ammonium and 2-oxoglutarate to L-glutamate, utilizing NADH as a cofactor and yielding NAD⁺ and H₂O as byproducts: Reaction 2: NH₄⁺ + 2-oxoglutarate + NADH → (GLDH) L-glutamate + NAD⁺ + H₂O. The rate at which NADH concentration decreases is directly correlated with the urea concentration in the sample and is monitored by measuring absorbance at 340 nm.

2.6.4. Measurement of serum creatinine levels as a marker of renal function

Creatinine concentrations in serum samples were analyzed quantitatively using a spectrophotometric method on the Roche cobas 8000 (Roche Diagnostics, Mannheim, Germany) automated-analyzer. In this kinetic colorimetric approach, which relies on the Jaffe method, creatinine interacts with picric acid under alkaline conditions to generate a yellow-orange chromophore (Reaction: Creatinine + Picric acid → [alkaline pH] yellow-orange complex). The complex’s absorbance was recorded at a wavelength of 505 nm, where the observed color intensity demonstrated a direct correlation with creatinine levels. A correction of −26 μmol/L (−0.3 mg/dL) was applied to serum/plasma creatinine measurements to eliminate the influence of nonspecific chromogenic substances, such as proteins and ketones.

2.6.5. Measurement of lactate levels in blood serum

Lithium heparin-coated syringes were utilized during the blood collection process to prevent coagulation. Lactate (mmol/L) quantification was performed through a fluorescence optical electrode method, using the ABL800 FLEX (Radiometer Medical ApS, Copenhagen, Denmark) blood gas analyzer developed by Radiometer.

2.7. Histopathological analysis

2.7.1. Histopathological procedure applied in renal tissue analysis

Rat kidneys obtained post-necropsy were subjected to fixation using a 10% neutral formalin solution to ensure tissue preservation. Following standard dehydration with graded alcohols and clearing in xylol, the tissue specimens were embedded in paraffin for sectioning. Sections of 5 µm thickness were mounted on poly-L-lysine-coated slides and stained with Hematoxylin & Eosin. Mononuclear cell infiltration, hemorrhage, and tubular degeneration were evaluated in six randomly selected fields using a semiquantitative scoring system: absent (0), mild (1), moderate (2), and severe (3).

2.8. Statistical analysis

Statistical evaluations were carried out utilizing IBM SPSS Statistics software (version 27.0; IBM Corporation, Armonk, NY, USA, 2020) on a Windows operating system. Figure preparation was performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, California, USA, 2018). Biochemical data are presented as the mean accompanied by the standard error of the mean (± SEM). The assumption of normality was assessed using the Shapiro-Wilk test (Supplementary File 1). If the normality assumption was satisfied, homogeneity of variances assumption was assessed using Levene’s test (Supplementary File 2). To evaluate the mean differences between the groups, one-way ANOVA or Welch’s ANOVA test was used if the normality assumption was met. Pairwise comparisons between groups were conducted using Tukey’s honestly significant difference (HSD) post-hoc test following a one-way ANOVA when the assumption of homogeneity of variances was met, or using the Games-Howell post-hoc test following Welch’s ANOVA when the assumption was violated. Histopathological findings were presented using median values along with their corresponding ranges (min–max). For the histopathological data obtained, intergroup differences were initially assessed using the nonparametric Kruskal–Wallis test, and pairwise comparisons were subsequently conducted with the Mann–Whitney U test to identify the source of significance. Findings were considered statistically significant when p-values were below 0.05.

3. Results

3.1. Biochemical findings

3.1.1. MDA levels in renal tissue

As demonstrated in Figure 1 and Table 1, administration of linezolid markedly increased MDA levels in renal tissue in the LZD group (7.30 ± 0.06) compared to the HG group (4.32 ± 0.09) (p < 0.001). While ATP treatment (6.28 ± 0.09) significantly attenuated the linezolid-induced elevation in renal MDA levels (p < 0.001), the reduction observed in the thiamin-treated group (7.36 ± 0.12) was not statistically significant. Moreover, renal MDA levels were significantly lower in the groups treated with TPP (TPLZD, 4.62 ± 0.07) and the ATP/thiamin/TPP combination therapy (ATTPL, 4.15 ± 0.04) compared to the LZD group (TPLZD: p < 0.001; ATTPL: p < 0.0001). Notably, MDA levels in the ATTPL group were statistically comparable to those observed in the HG group.

Figure 1.

Effects of linezolid, ATP, thiamin, TPP, and their combination on renal MDA and tGSH levels in rats.

Note: Results are presented as mean ± standard error of the mean (SEM).

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group; MDA: malondialdehyde; tGSH: total glutathione.

Table 1.

Assessment and p-value comparison of the effects of linezolid, adenosine triphosphate, thiamin, thiamin pyrophosphate and their combination on renal oxidative stress markers and blood serum BUN, creatinine and lactate levels in rats.

		Post hoc test p-values
Group comparisons	MDA*	tGSH**	SOD**	CAT*	BUN**	Cr*	Lactate*
HG vs. LZD	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
HG vs. ATLZD	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
HG vs. TLZD	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
HG vs. TPLZD	0.134	0.523	0.713	0.261	0.837	0.985	0.648
HG vs. ATTPL	0.717	0.988	0.484	0.999	0.997	1.000	0.999
LZD vs. ATLZD	<0.001	<0.001	<0.001	0.007	1.000	0.433	1.000
LZD vs. TLZD	0.994	0.673	0.984	1.000	1.000	1.000	0.893
LZD vs. TPLZD	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
LZD vs. ATTPL	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
ATLZD vs. TLZD	<0.001	<0.001	0.046	0.012	1.000	0.620	0.786
ATLZD vs. TPLZD	<0.001	0.001	<0.001	<0.001	<0.001	<0.001	<0.001
ATLZD vs. ATTPL	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
TLZD vs. TPLZD	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
TLZD vs. ATTPL	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
TPLZD vs. ATTPL	0.005	0.348	0.176	0.426	0.981	0.990	0.432
F value	320.889	250.080a	155.051a	78.553	237.461a	209.414	66.025
df (df1 / df2)	5 / 30	5 / 13.645	5 / 13.713	5 / 30	5 / 13.755	5 / 30	5 / 30
p	<0.001	<0.001b	<0.001b	<0.001	<0.001b	<0.001	<0.001

Notes: *One-way ANOVA was conducted to analyze group differences, followed by Tukey’s Honestly Significant Difference (HSD) test for post hoc comparisons.

**Welch ANOVA was conducted to analyze group differences, followed by Games-Howell test for post hoc comparisons.

^aAsymptotically F distributed.

^bWelch ANOVA p-values.

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group; MDA: malondialdehyde; tGSH: total glutathione; SOD: superoxide dismutase; CAT: catalase; BUN: blood urea nitrogen; Cr: creatinine; df: degree of freedom.

3.1.2. tGSH levels in renal tissue

tGSH levels were significantly reduced in the LZD group (4.04 ± 0.07) compared to the HG group (7.39 ± 0.11) (p < 0.001). ATP administration (5.33 ± 0.06) significantly suppressed the LZD-induced depletion (p < 0.001). Although a slight increase was observed in the thiamin-treated group (4.16 ± 0.05), it was not statistically significant. In contrast, tGSH levels were significantly elevated in both the TPLZD (7.00 ± 0.19) and ATTPL (7.49 ± 0.13) groups (p < 0.001 and p < 0.0001, respectively). Notably, the ATTPL therapy exerted a stronger protective effect, with tGSH levels statistically comparable to those in the HG group (Figure 1 and Table 1).

3.1.3. SOD and CAT activities in renal tissue

As illustrated in Figure 2 and Table 1, SOD activity was significantly reduced in the LZD group (3.65 ± 0.06) compared to the HG group (6.33 ± 0.08) (p < 0.001). ATP treatment (4.71 ± 0.07) significantly alleviated this reduction (p < 0.001), whereas the slight increase observed in the TLZD group (3.79 ± 0.22) did not reach statistical significance. In contrast, SOD activity was significantly elevated in both the TPLZD (6.13 ± 0.12) and ATTPL (6.71 ± 0.18) groups (p < 0.001 and p < 0.0001, respectively). Notably, enzyme activity in the ATTPL group was nearly restored to levels comparable to those observed in the HG group.

Figure 2.

Effects of linezolid, ATP, thiamin, TPP, and their combination on renal SOD and CAT activities in rats.

Note: Results are presented as mean ± standard error of the mean (SEM).

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group; SOD: superoxide dismutase; CAT: catalase.

A marked decline in CAT activity was observed in the LZD group (2.34 ± 0.10) relative to the HG group (4.80 ± 0.14) (p < 0.001). Administration of ATP (3.15 ± 0.16) partially reversed this reduction with statistical significance (p = 0.007), while the minor elevation detected in the TLZD group (2.38 ± 0.15) was not found to be significant. Conversely, CAT activity showed significant improvement in both the TPLZD (5.27 ± 0.09) and ATTPL (4.86 ± 0.22) groups when compared to the LZD group (p < 0.001 and p < 0.0001, respectively). Importantly, the enzymatic activity in the ATTPL group approached the levels recorded in the HG group, with no statistically significant difference between them (Figure 2 and Table 1).

3.1.4. Blood serum BUN levels

As presented in Figure 3 and Table 1, serum BUN concentrations were markedly elevated in the LZD group (172.17 ± 2.93) compared to the HG group (53.00 ± 2.49) (p < 0.001). Neither ATP (170.17 ± 6.17) nor thiamin (171.17 ± 8.16) administration resulted in a statistically significant reduction in BUN levels and substantial inter-individual variability in BUN measurements was noted within these groups. In contrast, BUN concentrations were significantly reduced in both the TPLZD (58.67 ± 4.13, p < 0.001) and ATTPL (55.00 ± 3.59, p < 0.0001) groups, with values in the ATTPL group nearly equivalent to those of the HG group.

Figure 3.

Effects of linezolid, ATP, thiamin, TPP, and their combination on blood serum BUN and creatinine levels in rats.

Notes: Results are presented as mean ± standard error of the mean (SEM).

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group; BUN: blood urea nitrogen.

3.1.5. Blood serum creatinine levels

Serum creatinine levels were markedly elevated in the LZD group (1.89 ± 0.03) compared to the HG group (0.71 ± 0.03) (p < 0.001). Treatment with ATP (1.78 ± 0.04) or thiamin (1.87 ± 0.05) did not produce significant changes in creatinine values. In line with findings from other biochemical markers, creatinine concentrations were markedly decreased in the TPLZD (0.75 ± 0.04, p < 0.001) and ATTPL (0.71 ± 0.05, p < 0.0001) groups, with values in the ATTPL group being statistically indistinguishable from those in the HG group (Figure 3 and Table 1).

3.1.6. Blood serum lactate levels

Lactate concentrations remained within the physiological range in the HG group (12.33 ± 0.67), while a statistically significant increase was detected in the LZD group (28.00 ± 1.07, p < 0.001). Compared to the LZD group, lactate levels in the ATLZD (28.33 ± 1.36) and TLZD (26.50 ± 1.12) groups showed no significant change. In contrast, the TPLZD group exhibited a notable reduction (14.50 ± 0.76, p < 0.001), and this effect was even more prominent in the ATTPL group (11.83 ± 0.87, p < 0.0001). Importantly, lactate values in the ATTPL group closely approximated those measured in the HG group (Figure 4 and Table 1).

Figure 4.

Effects of linezolid, ATP, thiamin, TPP, and their combination on blood serum lactate levels in rats.

Notes: Results are presented as mean ± standard error of the mean (SEM).

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group.

3.2. Histopathological findings

As illustrated in Figure 5A, renal tissues from the HG group exhibited normal histological architecture without any discernible pathological changes. In contrast, the LZD group showed pronounced tubular degeneration, along with extensive mononuclear cell infiltration and marked interstitial hemorrhage in the renal parenchyma (Figure 5B–C and Table 2). ATP treatment led to a reduction in the severity of tubular degeneration caused by linezolid, with only moderate damage observed (Figure 5D and Table 2). Furthermore, moderate levels of interstitial hemorrhage and mononuclear cell infiltration were noted in the renal tissue (Figure 5E and Table 2). Treatment with thiamin did not mitigate the histopathological alterations, including tubular degeneration and mononuclear infiltration, associated with linezolid (Figure 5F and Table 2). In contrast, administration of TPP resulted in a clear attenuation of linezolid-induced tubular degeneration, interstitial mononuclear cell infiltration, and hemorrhage (Figure 5G–H and Table 2). In the ATTPL group, which was administered the combined treatment of ATP, thiamin, and TPP, renal histology appeared normal, with no observable pathological alterations (Figure 5I and Table 2).

Figure 5.

(A) Normal histological appearance of renal tissue in the HG group, Gl: glomerulus; Tb: tubule (H&E, ×200). (B) Renal tissue from the LZD group showing severe tubular degeneration (*) and mononuclear cell infiltration (►) (H&E, ×200). (C) Renal tissue from the LZD group showing severe interstitial hemorrhage (➞) (H&E, ×200). (D) Renal tissue from the ATLZD group showing moderate tubular degeneration (*) (H&E, ×200). (E) Renal tissue from the ATLZD group showing moderate interstitial hemorrhage (➞) and mononuclear cell infiltration (►) (H&E, ×200). (F) Renal tissue from the TLZD group showing severe tubular degeneration (*) and mononuclear cell infiltration (►) (H&E, ×200). (G) Renal tissue from the TPLZD group showing mild tubular degeneration (*) and mononuclear cell infiltration (►) (H&E, ×200). (H) Renal tissue from the TPLZD group showing mild interstitial hemorrhage (➞) (H&E, ×200). (I) Normal histological appearance of renal tissue from the ATTPL group. GI: glomerulus; Tb: tubule (H&E, ×200).

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group.

Table 2.

Quantitative evaluation of histopathological alterations in rat kidney tissue.

	Histopathological grading data
Groups	Tubular degeneration	Interstitial mononuclear cell infiltration	Interstitial hemorrhage
Group comparisons	p-values
HG	0.00 (0.00–0.00)	0.00 (0.00–0.00)	0.00 (0.00–0.00)
LZD	3.00 (2.00–3.00)	3.00 (2.00–3.00)	3.00 (2.00–3.00)
ATLZD	2.00 (1.00–3.00)	2.00 (1.00–2.00)	2.00 (1.00–2.00)
TLZD	3.00 (2.00–3.00)	3.00 (2.00–3.00)	3.00 (2.00–3.00)
TPLZD	1.50 (0.00–2.00)	1.00 (0.00–1.00)	1.00 (0.00–2.00)
ATTPL	0.00 (0.00–2.00)	0.00 (0.00–1.00)	0.00 (0.00–1.00)
HG vs. LZD	0.001	0.002	0.001
HG vs. ATLZD	0.002	0.002	0.002
HG vs. TLZD	0.002	0.002	0.002
HG vs. TPLZD	0.007	0.019	0.021
HG vs. ATTPL	0.140	0.317	0.138
LZD vs. ATLZD	0.026	0.014	0.006
LZD vs. TLZD	0.523	1.000	0.523
LZD vs. TPLZD	0.005	0.003	0.003
LZD vs. ATPPL	0.003	0.002	0.002
ATLZD vs. TLZD	0.075	0.014	0.014
ATLZD vs. TPLZD	0.150	0.014	0.057
ATLZD vs. ATTPL	0.014	0.004	0.007
TLZD vs. TPLZD	0.011	0.003	0.005
TLZD vs. ATTPL	0.004	0.002	0.003
TPLZD vs. ATTPL	0.105	0.093	0.212

Notes: Median (minimum–maximum) values were used to represent the data. Statistical analysis was performed using the Mann–Whitney U test. For all groups n = 6.

Abbreviations: ATP: Adenosine triphosphate; TPP: thiamin pyrophosphate; HG: healthy group; LZD: linezolid alone group; ATLZD: ATP + linezolid group; TLZD: thiamin + linezolid group; TPLZD: TPP + linezolid group; ATTPL: ATP + thiamin + TPP + linezolid group.

4. Discussion

This study investigated the biochemical and histopathological consequences of linezolid-induced oxidative stress on kidney injury and lactic acidosis and evaluated the therapeutic potential of ATP, thiamin, TPP, and their triple combination. Our results demonstrated that ATP, TPP, and especially the triple combination markedly alleviated the biochemical and structural alterations caused by linezolid, whereas thiamin alone was insufficient.

Linezolid is an effective agent against multidrug-resistant Gram-positive bacteria [27], yet serious neurological [28], hematological [29], and metabolic [2,30] adverse effects have been reported with prolonged use (>14 days). Clinical cases have described acute interstitial nephritis during therapy [4,5], underscoring renal risk.

Although its nephrotoxic mechanism is not fully clarified, oxidative stress is central: linezolid disrupts mitochondrial activity [31], reducing ATP production and inducing lipid peroxidation [32,33]. MDA, a highly reactive oxidative molecule produced during LPO, serves as a significant biochemical marker for determining prolonged cellular damage and oxidative stress [33]. In our study, MDA levels were significantly elevated in the kidney tissue of the linezolid-treated group. Similarly, Kendir et al. demonstrated a significant increase in erythrocyte MDA levels following linezolid administration in rats [34]. Moreover, significantly elevated MDA levels have also been reported in patients receiving linezolid treatment and diagnosed with thrombocytopenia [35].

Living organisms possess an endogenous antioxidant defense system that prevents or limits the harmful effects of ROS [36]. During drug-induced oxidative stress, renal GSH reserves become depleted, reflecting impaired antioxidant capacity [37]. GSH, the major non-enzymatic intracellular antioxidant, detoxifies hydroxyl radicals and maintains redox balance [38]. Several studies have demonstrated the key role of GSH in scavenging ROS within renal tissue [34,38]. In this study, tGSH levels were measured to assess the antioxidant defense capacity of the kidney and were found to be significantly reduced in the linezolid-treated group, consistent with previous reports. Parallel to this decline, the enzymatic antioxidants SOD and CAT were also markedly decreased, indicating an impaired redox balance. Similarly, Wang et al. reported a dose-dependent reduction in SOD and CAT activities following linezolid administration in rats and showed that antioxidant co-treatment alleviated oxidative stress and prevented hematological toxicity [23]. These consistent findings further support the notion that oxidative stress is a major contributor to linezolid-induced renal damage.

BUN and serum creatinine are well-established biochemical markers of renal function. As end products of protein metabolism excreted by the kidneys, their elevation reflects impaired glomerular filtration and renal injury [39,40]. Both experimental and clinical studies have reported increased BUN and creatinine levels during linezolid-induced nephrotoxicity. Esposito et al. observed a persistent rise in serum creatinine in a transplant patient with linezolid-associated interstitial nephritis [4], whereas another rat study found only a mild elevation compared with controls [24]. In our study, however, both BUN and creatinine levels were significantly higher in linezolid-treated rats, clearly indicating nephrotoxic effects.

Lactic acidosis is a serious complication of linezolid therapy that can lead to life-threatening multiorgan failure [41]. Elevated lactate is a hallmark of mitochondrial dysfunction resulting from inhibition of respiratory chain complexes I and IV [42]. Prolonged use, particularly beyond six weeks, increases the risk through cumulative mitochondrial injury [30]. Several case reports have documented linezolid-induced lactic acidosis, often requiring renal replacement therapy for recovery [41,43,44]. Consistent with these findings, our study demonstrated markedly elevated serum lactate levels in linezolid-treated rats compared with controls, confirming the development of lactic acidosis.

The kidney is among the most energy-demanding organs, requiring continuous ATP production to sustain cellular and antioxidant functions [37]. This underscores the necessity of efficient energy production through ATP to support cellular functions [45]. Moreover, ATP is known to play critical roles not only in energy production but also in providing energy for ROS scavenging and antioxidant synthesis [46]. Linezolid disrupts mitochondrial ATP synthesis, resulting in oxidative stress and energy imbalance [9,47,48]. In this study, ATP treatment markedly reduced MDA levels and restored antioxidant parameters (tGSH, SOD, and CAT) toward normal values, supporting its protective effect against oxidative stress. Consistent with earlier reports, ATP also promotes glutathione synthesis and limits oxidative damage [20]. However, ATP administration did not significantly alter BUN, creatinine, or lactate levels, indicating limited impact on renal function and lactic acidosis.

Previous studies highlight the importance of correcting factors such as thiamin deficiency, hypoxemia, and hypoglycemia in managing linezolid-induced lactic acidosis [49]. Thiamin therapy has been reported to prevent lactic acidosis and alleviate other adverse effects of linezolid, including cytopenias and neuropathy [50]. However, in our study, thiamin did not significantly improve oxidative stress parameters or renal and lactate indices, contrasting with earlier findings. These results suggest that TPP, the active coenzyme form of thiamin, may provide a more effective approach for counteracting linezolid-induced mitochondrial dysfunction.

Several studies have demonstrated that TPP provides stronger protection against drug-induced oxidative injury than thiamin [51–53]. In our study, TPP markedly improved oxidative stress markers, renal function, and lactate levels. As a cofactor of pyruvate dehydrogenase, TPP facilitates the conversion of pyruvate to acetyl-CoA; its deficiency redirects pyruvate to lactate, leading to lactic acidosis [16]. Laus et al. similarly reported that TPP prevented lactate accumulation and enhanced aerobic capacity [17]. Moreover, the combined administration of ATP, thiamin, and TPP restored oxidative, renal, and metabolic parameters to near-control levels, indicating a synergistic protective effect through improved energy metabolism and antioxidant defense.

Histopathological findings supported the biochemical results, confirming that linezolid caused marked renal damage characterized by tubular degeneration and mononuclear cell infiltration [21,22,24,54]. ATP treatment reduced tubular injury to a moderate level but did not completely prevent interstitial hemorrhage, suggesting partial protection through improved energy metabolism [22]. Thiamin administration failed to significantly lessen tubular degeneration or inflammation, likely due to insufficient conversion to its active coenzyme form. In contrast, TPP markedly reduced both tubular and interstitial lesions, consistent with Dagel et al. who demonstrated its antioxidant protective effect against linezolid-induced renal injury [22]. Notably, kidneys from rats treated with the ATP, thiamin, and TPP combination appeared histologically normal, indicating that this therapy provided the most effective protection by preventing oxidative and structural damage.

Taken together, the response pattern observed across lactate and redox endpoints – namely the superior efficacy of TPP compared with thiamin, the ability of ATP to improve redox balance without normalizing lactate, and the capacity of ATTP to correct both metabolic axes – supports a model in which linezolid disrupts mitochondrial translation and ETC complexes [30,31,42]. This disruption lowers ATP production and increases ROS generation, as reflected by elevated MDA levels and reduced tGSH, SOD, and CAT activities. In this context, TPP likely mitigates hyperlactatemia by restoring PDH flux and reducing pyruvate diversion toward lactate [16–17], whereas ATP supplementation primarily enhances redox capacity and promotes glutathione biosynthesis [20,45,46]. The superior efficacy of the triple combination can thus be attributed to the concurrent reestablishment of PDH-dependent energy metabolism together with reinforcement of antioxidant defenses. Although further work with alternative dosing and clinically relevant models is warranted, these data nominate TPP – particularly in combination with ATP – as a promising strategy to counteract linezolid-induced renal injury and lactic acidosis.

5. Conclusions

This study evaluated the effects of ATP, thiamin, TPP, and their triple combination on linezolid-induced oxidative kidney injury and lactic acidosis in rats. Linezolid administration resulted in increased MDA levels, suppression of the antioxidant defense system, elevated BUN and creatinine levels, and lactate accumulation. ATP administration partially alleviated the oxidative stress and antioxidant depletion associated with linezolid in renal tissue; however, it failed to prevent hyperlactatemia and the increases in BUN and creatinine levels. Thiamin administration alone did not produce significant changes in the biochemical parameters altered by linezolid. In contrast, marked improvements were observed in the groups receiving TPP and the triple combination treatment. The triple combination was particularly effective in restoring oxidative stress markers, renal function indicators, and lactate levels to near-control values. These findings suggest that TPP may be a promising agent for mitigating linezolid-induced renal damage, and its combined use with ATP may exert an even more pronounced protective effect.

6. Limitations

The limited efficacy of thiamin against linezolid-induced nephrotoxicity suggests that thiamin pyrophosphokinase (TPK), the enzyme responsible for converting thiamin into its active form, TPP, may be inhibited by linezolid. However, this hypothesis could not be directly tested in the present study, as renal thiamin and TPP levels, TPK activity, and mitochondrial respiratory complex functions were not measured. Future research should address these parameters to determine whether TPK inactivation contributes to the observed effects. Moreover, to better characterize the inflammatory component of renal injury, subsequent studies should evaluate pro-inflammatory cytokines and their modulation by ATP, thiamin, and TPP. Further investigations including early renal injury biomarkers, gene and protein expression analyses, and apoptotic markers are also warranted to elucidate the molecular mechanisms underlying the protective effects identified in this study.

Funding Statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.