N-acetyl cysteine amide mitigates oxidative stress and apoptosis in a rat model of renal ischemia-reperfusion injury

Abstract

Renal ischemia–reperfusion (IR) injury is a major cause of acute kidney injury, in which oxidative stress (OS) and apoptosis play central roles. N-acetyl cysteine amide (NACA), a lipophilic derivative of N-acetylcysteine, exhibits improved cellular penetration and antioxidant activity. This study investigated the renoprotective effects of NACA in a rat model of renal IR injury. Twenty-eight female Wistar albino rats were randomized into four groups (n = 7): Control, IR, NACA + IR (100 mg/kg i.p., 30 min before ischemia), and IR + NACA (100 mg/kg i.p., immediately after ischemia). Following right nephrectomy, the left renal pedicle was clamped for 60 min and reperfused for 24 h. Serum renal function markers, kidney OS parameters, histopathological injury, and caspase-3 immunoreactivity were evaluated. Renal IR injury significantly increased serum blood urea nitrogen and creatinine levels and induced histopathological damage characterized by tubular dilatation, cast formation, and degeneration. Catalase (CAT) and superoxide dismutase (SOD) activities were significantly altered; malondialdehyde increased after IR and was reduced by NACA pretreatment, whereas myeloperoxidase and total glutathione did not differ significantly among groups. NACA pretreatment attenuated inflammatory cell infiltration, tubular dilatation, and caspase-3 immunoreactivity, while partially restoring CAT and SOD activity. Post-ischemic NACA administration was less effective, particularly in reducing apoptosis and inflammatory infiltration. NACA confers partial renoprotection against renal IR injury, with pretreatment providing superior efficacy. These findings highlight the importance of antioxidant timing and suggest NACA as a potential prophylactic strategy when renal ischemia is predictable.

Introduction

Renal ischemia-reperfusion injury (IRI) in rats is a complex process that occurs when blood supply to the kidneys is temporarily cut off and then restored. Renal IRI in rats effectively mimics acute and chronic kidney diseases and diabetes-related kidney damage. This model is crucial for understanding the systemic effects of kidney injury and for developing therapeutic strategies¹.

Oxidative stress (OS) is a critical factor in the pathogenesis of renal IRI, contributing to mitochondrial dysfunction, inflammation, and apoptosis. Understanding these mechanisms has led to the development of antioxidant therapies and identification of potential biomarkers, offering promising avenues for improving clinical outcomes in acute kidney injury (AKI). During ischemia-reperfusion (IR), OS results from an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense system, leading to lipid peroxidation, protein and DNA damage, and ultimately cell apoptosis and necrosis^{2, 3}. OS is closely linked with inflammation and apoptosis in renal IRI. It activates signaling pathways such as mitogen-activated protein kinases (MAPKs)/nuclear factor-kappa B (NF-κB), leading to increased expression of inflammatory cytokines and apoptotic markers^{3, 4}.

N-acetyl cysteine amide (NACA) does have antioxidant effects. It is a derivative of N-acetyl cysteine (NAC), which is widely recognized for its antioxidant properties. NAC serves as a precursor for glutathione (GSH), a critical antioxidant in cellular defense against OS⁵. Although NAC has been extensively investigated as an antioxidant therapy in renal IRI, its renoprotective efficacy remains inconsistent across experimental and clinical studies. These variable outcomes are largely attributed to NAC’s limited bioavailability, poor cellular and mitochondrial penetration, and rapid extracellular metabolism, which restrict its ability to counteract intracellular OS during reperfusion⁶. In contrast, NACA is a more lipophilic derivative of NAC that readily crosses biological membranes, resulting in improved intracellular and mitochondrial distribution⁷. Preclinical studies have demonstrated that NACA exerts stronger antioxidant and antiapoptotic effects than NAC in models of contrast-induced nephropathy, neurotoxicity and traumatic injury, primarily through enhanced suppression of OS and apoptotic signaling pathways^{8, 9, 10}. Despite these promising properties, the effects of NACA in renal IRI remain poorly characterized. Therefore, the present study aimed to investigate whether NACA confers superior renoprotection in a rat model of renal IRI and to evaluate the importance of administration timing on its antioxidant and antiapoptotic efficacy.

Materials and methods

Animals

This study utilized 28 female Wistar albino rats, aged 3 months and weighing between 216 and 355 g, sourced from the Inonu University Laboratory Animals Research Center. They were housed in a controlled environment with a temperature of 21 ± 2 °C and humidity of 60 ± 5%, under a 12:12 h light:dark cycle. Rats were provided with a regular chow pellet diet and unrestricted access to tap water. Randomization was employed to allocate animals to several experimental groups and to gather and process data, with analysis conducted by scientists unaware of the treatment groups. All experimental procedures were approved by the Inonu University Faculty of Medicine Experimental Animal Ethics Committee (Protocol No: 2015/A-35) and were carried out in accordance with relevant institutional and national guidelines and regulations. Reporting follows the ARRIVE 2.0 guidelines. Animals were sourced, housed, handled, anesthetized, and euthanized according to established standards to minimize suffering and reduce the number of animals used¹¹. A straightforward randomization method was employed to assign the rats to groups, therefore mitigating bias in the experimental procedure.

Chemicals

NACA (CAS number: 38520-57-9, Sigma Aldrich, St. Louis, MO, USA), ketamine (Ketasol 10% w/v injectable solution; Richter Pharma Ag, Wels, Austria), xylazine (XylazinBio 2% injectable solution, Bioveta PLC, Ivanovice na Hané, Czech Republic) and povidone-iodine 10% w/v (Batticon antiseptic solution, ADEKA Pharmaceuticals, İstanbul, Türkiye) were purchased.

Experimental design

Four groups were formed with 7 animals in each group and right nephrectomy was performed in each group. Nephrectomized animals were treated according to the experimental groups as follows;

Group 1-Control: In the control group, ischemia was not performed in the left kidney after right nephrectomy.

Group 2-IR: In this group, after right nephrectomy (Fig. 1A), left renal artery and vein were clamped together for 60 min of ischemia (Fig. 1B) and 24 h of reperfusion with surgical closure (Fig. 1C).

Fig. 1.

Schematic representation of the experimental model. (A) Right nephrectomy was performed in all groups. (B) Left renal artery and vein were clamped for 60 min to induce ischemia. (C) Reperfusion was established for 24 h following clamp removal.

Group 3-NACA + IR (100 mg/kg): In this group, NACA was administered at 100 mg/kg intraperitoneally (i.p.) 30 min before left renal IR after right nephrectomy and the group was left to reperfusion for 24 h with surgical closure.

Group 4-IR + NACA (100 mg/kg): In this group, NACA was administered at 100 mg/kg i.p. after 60 min of left renal ischemia after right nephrectomy and the group was left to reperfusion for 24 h with surgical closure.

The study by Cengiz et al. was referenced in the selection of the experimental renal IR model¹². The dosage of NACA were referenced from a previous study¹³. The abdominal region was desinfected with povidone-iodine 10% solution. Establishment of the experimental IR model and tissue harvesting and blood collection were performed under intraperitoneal 100 mg/kg ketamine and 10 mg/kg xylazine anesthesia. The rats were then euthanized by surgical exsanguination. Subsequently, kidney tissue and blood specimens were collected from rats, followed by histopathological and biochemical studies. The kidneys were promptly excised, decapsulated, and bisected into two equal longitudinal segments. One portion was preserved in formalin for histopathological evaluation, while the remaining tissues were maintained at −80 ˚C for biochemical investigation. Blood samples were obtained in tubes to assess blood urea nitrogen (BUN), creatinine (Cr) and albumin levels. Histopathological assessments (congestion, infiltration, caste formation, tubular dilatation, tubular degeneration, glomerulus diameter and caspase-3 reactivity) and biochemical analyses [catalase (CAT), superoxide dismutase (SOD), total GSH (tGSH), malondialdehyde (MDA), myeloperoxidase (MPO)] were conducted at the conclusion of the study protocol.

Biochemical analysis

Serum biochemical analysis

Serum concentrations of BUN, Cr, and albumin were analyzed at the Central Laboratory of Inonu University Turgut Özal Medical Center. BUN was measured using a urease–glutamate dehydrogenase enzymatic method (Roche Diagnostics, Mannheim, Germany)¹⁴, Cr was determined by the kinetic Jaffe method (Roche Diagnostics, Mannheim, Germany)¹⁵, and albumin levels were assessed by a bromocresol green colorimetric assay (Abbott Diagnostics, IL, USA). All analyses were carried out with an automated chemistry analyzer ¹⁶, in accordance with the manufacturers’ protocols.

Kidney tissue biochemical analysis

Kidney tissue was homogenized by adding 1/10 (w/v) phosphate-buffered saline (PBS) (pH 7.4) using an IKA-Werke T25 homogenizer under ice-cold conditions until complete tissue disruption was achieved. The homogenates were then sonicated four times for 15 s at 30 s intervals using a Sonics VCX130 sonifier. An aliquot of 500 µL from the homogenate was reserved for MDA measurement. Following sonication, samples were centrifuged at 10,000 rpm at + 4 °C for 10 min using a Nüve NF 800R microcentrifuge. The resulting supernatants were collected for enzyme activity assays and protein determination and stored at −80 °C until analysis.

Catalase activity (CAT)

CAT activity was measured in the supernatants according to the method of Lück¹⁷. CAT catalyzes the degradation of hydrogen peroxide (H₂O₂) into water and molecular oxygen. The rate of H₂O₂ decomposition was measured spectrophotometrically at 240 nm based on the decrease in H₂O₂ absorbance. In brief, 1000 µL of freshly prepared H₂O₂ solution was placed in a cuvette, and increasing volumes of supernatant starting from 30 µL were added depending on the working range. After gentle mixing, the absorbance change was recorded for 30 s at 240 nm using a Shimadzu 1601 UV–visible spectrophotometer (ɛ = 0.0396 cm² µmol⁻¹). Enzyme activity was calculated as units per milliliter from the optical density change.

Superoxide dismutase activity (SOD)

SOD activity, which reflects the dismutation of superoxide radicals, was determined according to the McCord method¹⁸. This assay is based on the inhibition by SOD of cytochrome-c reduction mediated by superoxide radicals generated in the xanthine–xanthine oxidase system.

Total glutathione determination (tGSH)

Total GSH levels were measured with minor modifications of the method described by Akerboom and Sies¹⁹. This method quantifies total GSH content by measuring reduced GSH generated from both reduced and oxidized GSH in tissues. Briefly, 1 mL phosphate buffer, 10 µL sample, 50 µL nicotinamide adenine dinucleotide phosphate (NADPH) solution, 20 µL 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) solution, and 20 µL GSH reductase were added to a 1 mL cuvette. After incubation for 15 min, absorbance was measured at 412 nm against a blank.

Protein determination

Protein concentrations were determined using the Bradford method²⁰. Samples were mixed with Bradford reagent, and protein levels were quantified according to the appropriate dilution factors using a standard curve generated with albumin. Results were expressed as mg/mL.

Determination of lipid peroxidation (MDA)

MDA levels in kidney tissue homogenates were determined using the method of Uchiyama and Mihara²¹. Lipid peroxidation results in the formation of MDA, which reacts with thiobarbituric acid (TBA) at 100 °C to form a pink-colored complex. Briefly, 0.025 g of tissue was homogenized in 500 µL PBS and sonicated for a total of 60 s (four cycles of 15 s). The homogenate was transferred to heat-resistant glass tubes, mixed with 2000 µL trichloroacetic acid–thiobarbituric acid–hydrochloric acid (TCA–TBA–HCl) reagent, and incubated in a boiling water bath at 100 °C for 30 min. After centrifugation at 15,000 rpm for 10 min, the supernatant was collected and absorbance was measured at 532 nm. MDA levels were expressed as nmol/g wet tissue and nmol/mg protein.

Myeloperoxidase (MPO) detection

MPO activity was determined as an indicator of neutrophil infiltration. Briefly, 0.1 g of tissue was homogenized in 1 mL phosphate buffer and centrifuged at 15,000 g for 15 min. The supernatant was discarded, and the pellet was resuspended in 500 µL hexadecyltrimethylammonium bromide (HETAB) solution. Samples underwent three cycles of freezing–thawing followed by sonication. After final centrifugation at 15,000 g for 15 min, the supernatant was collected. Aliquots of 25 µL were transferred to 96-well plates, mixed with 200 µL reaction mixture, and absorbance was measured spectrophotometrically at 460 nm at 3 and 5 min²². MPO activity was expressed as U/g wet tissue.

Histopathological analyses

The kidney tissue taken at the end of the experiment was fixed in 10% formaldehyde. After tissue tracing, 4–5 μm thick sections were taken from the paraffin blocks prepared. Hematoxylin-eosin (H-E) staining method was applied to the sections to determine the general morphologic structure.

Kidney sections were examined for congestion, infiltration, tubular degeneration, tubular dilatation, and cast formation. Damage was scored as 0 (no change), 1 (mild damage), 2 (moderate damage) and 3 (severe damage) according to severity and 10 randomly selected areas were evaluated. In addition, the diameter of 20 randomly selected glomeruli was measured in each section. Analyses were performed with a Leica DFC-280 research microscope using the Leica Q Win Image Analysis System (Leica Micros Imaging Solutions Ltd., Cambridge, UK).

Immunohistochemical analysis

For immunohistochemical analysis, deparaffinized and rehydrated sections were placed in a pressure cooker and boiled in 0.01 M citrate (pH 6.0) for 15–20 min. The sections were treated with 3% H₂O₂ for 12 min to block endogenous peroxidase enzyme activity. Protein block (ultra V block) was applied to the sections washed with PBS for 5 min. The sections were then incubated with primary antibody (Caspase-3, Thermo Scientific) at 37 ˚C for 60 min. The tissues were washed with PBS and treated with biotinylated secondary antibody at 37 ˚C for 10 min. After this process, the sections were incubated with streptavadin peroxidase at 37 ˚C for 10 min. The chromogen-treated sections were then stained with hematoxylin and covered with water-based sealer.

Staining was scored semiquantitatively based on the extent (0: 0–25%, 1: 26–50%, 2: 51–75%, 3: 76–100%) and severity (0: absent, + 1: mild, + 2: moderate, + 3: severe) of immunoreactivity. Total staining score was obtained by calculating prevalence x severity²³.

Statistical analyses

Statistical analyses were performed using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Data distribution was assessed using the Shapiro–Wilk normality test. Normally distributed data were expressed as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test for multiple comparisons. Non-normally distributed data were expressed as median (minimum–maximum) and analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons post-hoc test when appropriate. Adjustments for multiple comparisons were applied within the respective post-hoc analyses. A p value < 0.05 was considered statistically significant.

Results

Body and kidney weights of rats

After completion of the experimental protocol, the body and kidney weights of the rats are presented in Fig. 2.

Fig. 2.

Comparison of final body and kidney weights across experimental groups. (a) There is a statistically significant difference compared to the control group (p < 0.05). (b) There is a statistically significant difference compared to the IR group (p < 0.05).

Serum biochemical analysis

The results for serum BUN, Cr, and albumin parameters are presented in Fig. 3. In the renal IR model, BUN levels were significantly higher in the IR group compared to the control group (p < 0.05), but no significant difference was observed between the NACA groups and the IR group. Cr levels were significantly higher in the IR group compared to the control group (p < 0.05). In the NACA groups, these levels approached the control group, but there was no significant difference between them and the IR group. No significant correlation was observed between the groups in albumin results.

Fig. 3.

Comparison of serum renal function markers (BUN, creatinine and albumin) among experimental groups. BUN: blood urea nitrogen. (a) There is a statistically significant difference compared to the control group (p < 0.05). (b) There is a statistically significant difference compared to the IR group (p < 0.05).

Kidney tissue biochemical analysis

Biochemical analyses performed on kidney tissue are presented in Table 1; Fig. 4. There were statistically significant differences between groups in CAT, SOD, and MDA parameters related to OS in kidney tissue, while there were no statistically significant differences between groups in tGSH, and MPO parameters.

Table 1.

Renal OS profiles and antioxidant response in experimental IRI.

	CAT (U/mg protein)	SOD (U/mg protein)	tGSH (nmol/mg protein)	MDA (nmol/g wet tissue)	MPO (U/g wet tissue)
Control	153.3 ± 16.2	45.4 ± 1.1	5.0 ± 0.6	7.8 ± 1.8	0.93 ± 0.51
IR	137.8 ± 10.9	37.1a ± 2.0	4.9 ± 0.3	11.8a ± 1.8	0.33 ± 0.11
NACA + IR	106.1a, b ± 7.1	43.8b ± 4.2	5.2 ± 1.0	6.4b ± 2.0	0.94 ± 0.67
IR + NACA	135.3c ± 16.9	40.1a, c ± 3.3	5.2 ± 0.9	10.3 ± 2.6	0.82 ± 0.44

CAT: catalase, SOD: superoxide dismutase, tGSH: total glutathione, MDA: malondialdehyde, MPO: myeloperoxidase.

^aThere is a statistically significant difference compared to the control group (p < 0.05).

^bThere is a statistically significant difference compared to the IR group (p < 0.05).

^cThere is a statistically significant difference compared to the NACA + IR group (p < 0.05).

Fig. 4.

Kidney tissue OS parameters in control, IR, and NACA-treated rats. CAT: catalase, SOD: superoxide dismutase, tGSH: total glutathione, MDA: malondialdehyde, MPO: myeloperoxidase. (a) There is a statistically significant difference compared to the control group (p < 0.05). (b) There is a statistically significant difference compared to the IR group (p < 0.05). (c) There is a statistically significant difference compared to the NACA + IR group (p < 0.05).

Histopathological findings

In the control group, congestion was observed in the interstitial area, and mild degenerative changes were observed in the tubules. In the sections belonging to the IR group, congestion was observed similar to the control group. At the same time, mild inflammatory cell infiltration was observed between the tubules and around the vessels. The most prominent changes in this group were tubular dilatation, cast formation, and tubular degeneration. Due to the dilation developing in the tubules, flattening of tubule cells was observed, while hydropic changes and shedding in the cells of some tubules were noteworthy (Fig. 5). The difference between the control group and the IR group in terms of these histopathological changes was found to be statistically significant (p < 0.05).

Fig. 5.

Control Group; thin arrows indicate congestion. IR Group; infiltration (stars), cast formation (thick arrows), tubular dilatation (dashed arrows), and tubular degeneration (arrowheads) are observed. H&E x20.

On the other hand, in the NACA + IR group, congestion, cast formation, and tubular degeneration continued similarly to the IR group (p > 0.05), while infiltration and tubular dilatation were found to be statistically significantly reduced compared to the IR group (p < 0.05). Except for inflammatory cell infiltration, the NACA + IR and IR + NACA groups were statistically similar to each other (p > 0.05) (Fig. 6).

Fig. 6.

NACA + IR Group; congestion (thin arrows), cast formation (thick arrows), and tubular degeneration (arrowheads) are observed. IR + NACA Group; in addition to the histological changes observed in the NACA + IR group, infiltration (stars) is observed in this group. H&E x20.

Glomerular diameters were presented in Table 2.

Table 2.

Histopathological scores and caspase-3 immunoreactivity results (median (min-max)) and glomerular diameters (mean ± SD) of the groups.

	Control	IR	NACA + IR	IR + NACA
Congestion	1.0 (0.0–2.0)	1.0 (0.0–3.0)	1.0 (0.0–3.0)	1.0 (0.0–3.0)
Infiltration	0.0 (0.0–1.0)	0.0 (0.0–3.0)	0.0 (0.0–2.0)	0.0 (0.0–3.0)
Cast formation	0.0 (0.0–0.0)	0.0 (0.0–2.0)	0.0 (0.0–3.0)	0.0 (0.0–3.0)
Tubular dilatation	0.0 (0.0–2.0)	2.0 (0.0–3.0)	1.0 (0.0–3.0)	1.0 (0.0–3.0)
Tubular degeneration	0.0 (0.0–2.0)	1.0 (0.0–3.0)	2.0 (0.0–3.0)	2.0 (0.0–3.0)
Glomerular diameter (µm)	97.86 ± 12.46	101.09 ± 14.56	100.68 ± 15.95	97.46 ± 13.52
Caspase-3 reactivity	0.0 (0.0–1.0)	1.0 (0.0–2.0)	0.0 (0.0–1.0)	1.0 (0.0–2.0)

Immunohistochemical findings

Caspase-3 immunoreactivity was observed in the cytoplasm of distal tubule cells (Tables 2 and 3; Fig. 7). Immunoreactivity was found to be higher in the IR group compared to the control group (p < 0.05). When compared with the IR group, a statistically significant decrease in caspase-3 immunoreactivity was observed in the NACA + IR group, whereas a statistically significant increase was detected in the IR + NACA group (p < 0.05).

Table 3.

p values.

	Congestion	Infiltration	Cast formation	Tubular dilatation	Tubular degeneration	Glomerular diameter	Caspase-3 reactivity
Control-IR	0.332	0.001	0.001	0.001	0.001	0.335	0.004
IR - NACA + IR	0.320	0.009	0.860	0.004	0.640	1.000	0.013
IR - IR + NACA	0.675	0.919	0.416	0.001	0.147	0.242	0.002
NACA + IR - IR + NACA	0.691	0.018	0.351	0.725	0.252	0.371	0.001

Fig. 7.

Caspase-3 immunoreactivity observed in distal tubule cells in each group (arrows). x20.

Discussion

IRI is one of the leading causes of AKI, with high morbidity and mortality in clinical practice. It is characterized by the sudden interruption of blood flow followed by reperfusion, which paradoxically exacerbates tissue damage through OS, inflammation, and apoptosis^{1, 2}. In our study, we demonstrated that administration of NACA conferred partial protection against renal IRI in rats. Specifically, NACA pretreatment attenuated inflammatory cell infiltration, tubular dilatation, and caspase-3 immunoreactivity, while modulating antioxidant enzyme activities such as CAT and SOD. These findings highlight the potential role of NACA as a therapeutic antioxidant, particularly in conditions where renal ischemia can be anticipated, such as transplantation or major surgery.

The superior efficacy of NACA pretreatment compared with post-ischemic administration can be explained by the temporal dynamics of IRI. A pronounced burst of ROS occurs immediately upon reperfusion, leading to rapid mitochondrial dysfunction, lipid peroxidation, and activation of apoptotic signaling pathways^{24, 25}. Antioxidants administered after this early oxidative surge may have limited capacity to reverse established molecular damage. Highly reactive species like hydroxyl radical, peroxynitrite and hypohalous acids react with proteins, lipids and DNA almost instantly; they are too fast to be scavenged effectively by exogenous small‑molecule antioxidants once formed. Biomolecules generally react faster with ROS than do low‑molecular‑weight antioxidants, so antioxidants added after the burst struggle kinetically and thermodynamically to compete with damage reactions^{26, 27}.

NACA, owing to its increased lipophilicity and enhanced cellular and mitochondrial penetration compared with NAC, likely requires sufficient tissue distribution before reperfusion to effectively neutralize the initial oxidative burst^{7, 9, 28}. When administered as a pretreatment, NACA is already present within renal tubular cells at the onset of reperfusion, allowing early suppression of OS and downstream apoptotic signaling, as reflected by reduced caspase-3 immunoreactivity and improved histological outcomes in our study. In contrast, post-ischemic administration may occur after critical oxidative and inflammatory cascades have been initiated, thereby attenuating its protective efficacy. This timing-dependent effect underscores the importance of antioxidant availability during the early reperfusion phase, when OS–mediated injury is most intense and potentially irreversible.

Renal IRI consistently leads to significant increases in serum BUN and Cr levels, reflecting acute impairment of renal function. These elevations are observed as early as a few hours post-injury, peaking around 24 h, and are widely used as markers of renal dysfunction in both animal models and clinical settings. The magnitude of these changes correlates with the severity and duration of ischemia^{29, 30}. Our results are consistent with earlier studies showing that renal IRI induces marked elevations in serum BUN and Cr levels, together with histopathological alterations such as tubular degeneration, cast formation, and inflammatory infiltration^{30, 31}. Histological examination after IRI reveals classic features of acute tubular injury. Loss of brush border, tubular epithelial cell swelling, vacuolization, and necrosis are common findings. Tubular lumens often contain proteinaceous casts and cellular debris, indicating severe tubular damage. There is prominent infiltration of inflammatory cells, including neutrophils and monocytes, into the interstitium and tubules, contributing to further injury. Edema, tubular dilation, and congestion are also frequently reported^{29, 30, 32, 33}. Williams et al. demonstrated that although serum Cr levels normalize within days after reperfusion, histopathological damage may persist, indicating that tissue-level injury is more sustained. The peak in BUN and Cr levels typically coincides with the most severe histopathological changes, especially at 24 h post-ischemia. While renal function markers may normalize over time, histopathological evidence of injury can persist longer³⁰. In our study, biochemical parameters partially improved with NACA treatment, but histopathological and immunohistochemical markers provided more sensitive evidence of its protective action, especially when given before ischemia. This discrepancy between biochemical and histological recovery is in line with earlier reports, suggesting that functional renal parameters. Although NACA pretreatment significantly reduced inflammatory cell infiltration, tubular dilatation, and caspase-3 immunoreactivity, it did not significantly improve cast formation or tubular degeneration. These histopathological features are generally considered markers of more advanced and structural tubular injury, reflecting epithelial cell loss, luminal obstruction, and impaired tubular integrity^{34, 35}. Once established, such damage may be less amenable to short-term antioxidant intervention, particularly within the early 24-hour reperfusion period examined in this study. Therefore, the absence of improvement in cast formation and tubular degeneration suggests that NACA primarily mitigates early OS–driven and apoptotic processes rather than fully reversing established structural injury. This pattern supports the interpretation of NACA as providing partial renoprotection rather than complete histopathological recovery.

OS plays a pivotal role in renal IRI, leading to lipid peroxidation, protein oxidation, and DNA damage^{2, 3}. Markers such as MDA and TBA-reactive substances are significantly elevated after renal IRI, indicating extensive lipid peroxidation. Antioxidant treatments reduce these markers and tissue damage^{12, 36, 37, 38}. Increased protein carbonyl content and nitrosylation are observed post-IRI, reflecting protein oxidation. Antioxidants and specific inhibitors can ameliorate these changes^{39, 40, 41}. DNA damage response markers are upregulated after IRI, and interventions that reduce OS also decrease DNA damage and subsequent cellular senescence^{42, 43}.

Previous studies have reported that mitochondrial dysfunction is a central event, as ischemia triggers excessive production of ROS and nitric oxide (NO), further amplifying tissue injury³¹. In our study, IR significantly reduced SOD activity, while NACA pretreatment restored it to near-control levels. This aligns with the results of Granata et al., who emphasized that reinforcement of endogenous antioxidant defenses can mitigate reperfusion-associated oxidative bursts⁴⁴.

Moreover, our finding that CAT activity was altered by NACA pretreatment suggests that NACA acts at multiple levels of antioxidant defense. Research in a rat model of hepatic IRI shows that CAT activity increases significantly after IR. When rats were pretreated with NAC, CAT activity remained elevated, similar to the untreated IR group, indicating that NAC does not suppress the ischemia-induced increase in CAT activity. Instead, NAC pretreatment helps maintain antioxidant defenses, as seen by preserved GSH levels and reduced markers of oxidative damage, but does not specifically reduce or normalize CAT activity after ischemia⁴⁵.

MDA is a marker of lipid peroxidation and OS. In the present study, MDA levels were significantly increased in the IR group compared with control and were significantly reduced by NACA pretreatment (p < 0.05). MPO activity reflects neutrophil infiltration and inflammation. NAC administration consistently decreases MPO activity in IRI across different organs, demonstrating anti-inflammatory effects^{46, 47, 48, 49}. NACA, a derivative of NAC, also significantly lowers MDA levels in traumatic brain injury models, suggesting a similar effect in ischemic conditions. NACA has not been directly studied for MPO in ischemia, but its antioxidant and antiapoptotic effects are well-documented⁵⁰. In contrast, MPO activity did not reach statistical significance among groups, despite numerical differences, indicating that the NACA regimens did not measurably modify MPO at the 24-h reperfusion time point in this model. This emphasizes that antioxidant monotherapy may not be sufficient, and combinatorial strategies targeting ferroptosis, apoptosis, and inflammation may be more effective. The lack of change in tGSH levels across groups indicates preservation of the global GSH pool, despite alterations in specific antioxidant enzymes. This pattern is compatible with a scenario in which early enzymatic antioxidant responses (SOD/CAT) shift without producing a detectable depletion of the tGSH pool at this single 24 h endpoint.

NAC is a widely studied antioxidant with proven clinical applications, especially in acetaminophen toxicity. Its mechanism includes replenishing intracellular GSH, breaking disulfide bonds, and modulating thiol redox balance^{5, 51, 52}. Our results extend this observation to renal IRI, suggesting that NACA may serve as a more potent alternative to NAC for renal protection. A direct comparison in a rat model of contrast-induced nephropathy found that NACA provided superior renal protection compared to NAC at equimolar doses. NACA more effectively reduced serum Cr, BUN, and biomarkers of AKI, and it better preserved renal histology. The enhanced effect is attributed to NACA’s improved membrane permeability and antioxidant capacity, which allow for greater tissue penetration and efficacy in reducing OS and apoptosis in renal cells. NACA more effectively upregulates thioredoxin-1, inhibits the apoptosis signal-regulating kinase1/p38 MAPK pathway, and suppresses OS and apoptosis, key mechanisms in renal injury⁵³. NACA’s amide modification increases lipophilicity and membrane permeability, overcoming NAC’s limitations of low bioavailability and tissue penetration. While NAC is widely used, its clinical benefit for renal protection is inconsistent, and guidelines remain divided. NACA’s superior preclinical performance suggests it could address these gaps, though human studies are needed^{7, 53}.

There is currently no direct research evidence in the provided literature that NACA reduces caspase-3 activity or inhibits apoptosis. However, the role of caspase-3 as a central executioner in apoptosis is well established, and its inhibition is a recognized strategy for reducing apoptosis in OS-related conditions. The ability of NACA to reduce caspase-3 activity further supports its potential role in inhibiting apoptosis, a downstream event of OS⁴. These advantages may make NACA a more attractive therapeutic candidate in clinical settings where rapid and efficient antioxidant action is required. Furthermore, caspase-3 immunoreactivity, was markedly increased in the IRI group but attenuated with NACA pretreatment. Interestingly, NACA administration after ischemia was less effective, underscoring the importance of early intervention to intercept the initial burst of oxidative and inflammatory mediators upon reperfusion.

IR not only triggers OS but also activates inflammatory cascades, primarily via NF-κB and MAPK signaling pathways^{3, 4}. In our study, tubular infiltration of inflammatory cells was significantly reduced by NACA pretreatment. This is consistent with Hu et al., who reported that suppression of toll-like receptor-4/NF-κB signaling mitigated OS and inflammatory responses in IRI⁴.

From a translational perspective, our findings have important implications for clinical scenarios such as kidney transplantation, partial nephrectomy, or vascular surgery, where renal ischemia can be predicted. Preemptive administration of NACA may protect renal tissue during reperfusion, thereby reducing the risk of delayed graft function or AKI. However, the incomplete normalization of biochemical parameters such as MDA and MPO in our study indicates that NACA alone may not fully protect against IRI. Future studies should therefore explore combinational approaches, such as NACA with ferroptosis inhibitors, mitochondrial stabilizers, or anti-inflammatory drugs, to achieve more comprehensive protection^{31, 44}.

Several limitations of the present study should be acknowledged. First, we assessed only the early phase (24 h) of reperfusion, whereas long-term functional and structural outcomes remain unexplored. Second, only one dose of NACA (100 mg/kg) was tested; dose-response studies are required to optimize therapeutic efficacy. Third, while we demonstrated reductions in caspase-3 activity, other apoptotic and necroptotic pathways were not investigated. Advanced molecular analyses including ferroptotic markers, mitochondrial bioenergetics, and transcriptomic profiling could further elucidate NACA’s mechanism of action. Finally, extrapolation to humans requires caution, as metabolic and pharmacokinetic profiles differ significantly between rodents and humans.

Conclusion

In conclusion, this study demonstrates that NACA provides partial renoprotection in renal IRI by modulating OS enzymes, attenuating histopathological injury, and reducing apoptotic signaling. Its beneficial effects were more pronounced when administered prior to ischemia, highlighting the importance of timing in antioxidant therapy. Although NACA did not completely normalize all biochemical markers, its unique pharmacological properties compared with NAC suggest promising clinical potential. In this context, the designation of partial renoprotection specifically reflects the selective modulation of OS parameters, including antioxidant enzymes (SOD and CAT) and lipid peroxidation (MDA) together with histopathological and antiapoptotic improvements, rather than a global normalization of all OS markers such as MPO, and tGSH.

Future studies should focus on long-term outcomes, combination therapies, and translational models to fully establish NACA as a therapeutic option for IR–associated kidney injury.

Author contributions

Onural Ozhan: Conceptualization, Software, Visualization, Data curation, Writing—Original draft preparation, Writing-Reviewing and Editing. Cihan Ekici: Supervision, Investigation, Data curation, Validation, Investigation, Methodology. Burhan Ates: Investigation, Methodology, Visualization. Azibe Yildiz: Investigation, Methodology, Visualization. Sevgi Balcioglu: Investigation, Methodology, Visualization. Nigar Vardi: Investigation, Methodology, Visualization. Hakan Parlakpinar: Supervision, Writing—Reviewing and Editing.

Funding

The research leading to these results received funding from the Scientific and Technological Research Council of Türkiye (TÜBİTAK) under Grant Agreement No:1919B011503666.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All experimental procedures were approved by the Inonu University Faculty of Medicine Experimental Animal Ethics Committee (Protocol No: 2015/A-35) and were carried out in accordance with relevant institutional and national guidelines and regulations. Reporting follows the ARRIVE 2.0 guidelines. Animals were sourced, housed, handled, anesthetized, and euthanized according to established standards to minimize suffering and reduce the number of animals used ¹¹.

Consent to participate

Not applicable.

Consent for publication

Not applicable.