Nephroprotective Effects of Post-contrast N-Acetylcysteine and Montelukast Treatment in a Rat Model of Contrast-induced Acute Kidney Injury

Summary

This protocol presents a rat model of contrast-induced acute kidney injury and evaluates the post-contrast administration of N-acetylcysteine and montelukast, separately and in combination. The treatment regimens were associated with less severe renal dysfunction, oxidative stress, inflammation, and histopathological injury, although combined treatment did not consistently outperform monotherapy.

Abstract

Contrast-induced acute kidney injury (CI-AKI) is an important complication of iodinated contrast exposure in which oxidative stress and inflammatory processes contribute to renal damage. This study established an experimental rat model of CI-AKI and examined the effects of treatment initiated after contrast administration with N-acetylcysteine (NAC), montelukast, or a combination of both. Adult male Wistar rats underwent water deprivation followed by sequential intravenous administration of Nω-nitro-L-arginine methyl ester (L-NAME), indomethacin, and iohexol. Treatment was initiated 1 h after completion of iohexol administration, approximately 1.5 h after L-NAME administration, and was continued once daily for 3 consecutive days. Renal function indices, oxidative stress parameters, inflammatory cytokines, renal injury biomarkers, and kidney histopathology were evaluated. The untreated CI-AKI group exhibited pronounced renal dysfunction, an unfavorable oxidative stress and inflammatory profile, and substantial histopathological damage. Compared with the untreated CI-AKI group, animals receiving NAC, montelukast, or the combined regimen generally showed improved biochemical and histopathological findings. Glutathione (GSH) levels were highest, and mean histopathological injury scores were numerically lowest in the combination group; however, combined treatment did not consistently outperform monotherapy across the assessed outcomes. This protocol provides a controlled preclinical framework for evaluating post-contrast interventions in CI-AKI, and the findings warrant further experimental investigation of NAC and montelukast without establishing clinical efficacy or a combination-specific advantage.

Introduction

Acute kidney injury (AKI) frequently complicates the course of hospitalized patients and contributes to increased morbidity, mortality, and healthcare resource use. As iodinated contrast media are routinely employed in diagnostic imaging and interventional procedures, deterioration of kidney function following contrast exposure has become an important clinical concern. Current nomenclature distinguishes contrast-associated acute kidney injury (CA-AKI), which encompasses any episode of AKI occurring after contrast administration, from contrast-induced acute kidney injury (CI-AKI), in which renal injury is causally attributed to the contrast agent. In controlled experimental models, where contrast exposure can be directly linked to renal damage, the term CI-AKI therefore remains widely used^1,2.

Patients with chronic kidney disease, diabetes mellitus, advanced age, heart failure, or other cardiovascular comorbidities are particularly susceptible to CI-AKI. Kidney function recovers in many affected patients; however, persistent renal impairment, the need for renal replacement therapy, and an increased risk of death may occur in more severe cases. CI-AKI is also associated with longer hospitalization and greater use of healthcare resources. These adverse consequences underscore the continuing need to develop effective preventive and therapeutic approaches for contrast-related renal injury^1,3,4,5.

The development of CI-AKI involves several interrelated processes, including altered renal perfusion, medullary oxygen imbalance, oxidative stress, inflammation, and direct tubular toxicity. Contrast exposure shifts intrarenal vascular tone toward vasoconstriction while weakening vasodilatory responses, thereby reducing renal blood flow. The outer medulla is particularly vulnerable because oxygen availability in this region is already limited under physiological conditions. A further decline in oxygen delivery after contrast administration may therefore precipitate hypoxic tubular injury^2,6,7.

Renal hypoxia following contrast exposure is accompanied by increased generation of reactive oxygen species (ROS), which contributes substantially to kidney injury. When ROS production exceeds endogenous antioxidant capacity, oxidative damage may develop in cellular lipids, proteins, and DNA, while inflammatory signaling is simultaneously enhanced. In both experimental and clinical settings, contrast administration has been associated with increased oxidative stress parameters and impaired antioxidant defenses. Molecular pathways implicated in this response include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase–dependent radical formation and activation of nuclear factor kappa-B (NF-κB)^1,6,8,9.

The contribution of inflammation indicates that CI-AKI cannot be explained by hemodynamic disturbance alone. Recent evidence has also implicated ferroptosis, nitrosative stress, and neutrophil extracellular traps in renal injury following contrast exposure. Recognition of these additional mechanisms has broadened the range of molecular targets being investigated for the prevention and treatment of CI-AKI¹.

Serum creatinine and urine output are commonly used to identify CI-AKI, but both may change only after renal injury has already developed. This limitation has encouraged the evaluation of earlier indicators, including kidney injury molecule-1 (KIM-1), which reflects tubular damage, and cystatin C, which is associated with changes in glomerular filtration. Evidence from experimental and clinical studies indicates that alterations in these biomarkers may become detectable during the early phase of CI-AKI and therefore support earlier assessment of renal injury^9,10,11.

Advances in understanding the pathophysiology of CI-AKI have encouraged the evaluation of pharmacological interventions directed at oxidative and inflammatory pathways. Adequate hydration remains the principal preventive measure, whereas the reported benefits of drug-based approaches have varied across studies. Consequently, no pharmacological treatment has yet demonstrated sufficient consistent efficacy to be adopted as a standard clinical intervention for CI-AKI^1,4,12,13.

N-acetylcysteine (NAC) has been widely evaluated as a pharmacological approach for limiting CI-AKI. Its proposed renoprotective effects are related to the scavenging of reactive species and the restoration of glutathione reserves. Early clinical support came from a landmark study of patients with chronic kidney disease, in which NAC reduced the incidence of contrast-related renal dysfunction¹⁴. Later clinical trials and evidence syntheses, however, produced inconsistent findings, with some studies reporting benefit and others showing no meaningful clinical effect. The effectiveness of NAC for CI-AKI, therefore, remains uncertain^12,15,16,17.

Montelukast selectively blocks the cysteinyl leukotriene receptor-1 and has long been used to treat asthma and allergic disorders. Experimental evidence also indicates that this agent exerts anti-inflammatory, antioxidant, and anti-apoptotic effects. Across different models of renal injury, montelukast administration has been associated with reduced lipid peroxidation, strengthened endogenous antioxidant defenses, less severe histopathological damage, and attenuation of kidney injury^{18,19,20,21,22}.

Evidence specifically examining montelukast in CI-AKI remains limited. Available experimental findings suggest that its renal effects may involve modulation of oxidative and inflammatory signaling. Reduced expression of NADPH oxidase 4 (NOX4), p22phox, and NF-κB has been proposed as one mechanism through which montelukast may attenuate contrast-related renal injury⁹.

NAC and montelukast influence partially distinct but overlapping pathways involved in renal injury. NAC primarily supports antioxidant defense, whereas montelukast has both anti-inflammatory and antioxidant actions. Concurrent administration of these agents may therefore affect multiple components of CI-AKI pathophysiology; however, whether their interaction produces an additive or synergistic benefit has not been established in experimental models^{18,19,20,21,22}.

Previous investigations have predominantly evaluated interventions administered before contrast exposure, whereas evidence concerning treatments initiated after exposure remains scarce. In particular, post-contrast administration of montelukast together with NAC has received limited investigation in experimental CI-AKI. The present study therefore evaluated montelukast and NAC, administered after contrast exposure either separately or in combination, with respect to renal function, oxidative stress parameters and antioxidant defenses, renal injury and inflammatory biomarkers, and histopathological alterations in a rat model of CI-AKI.

Protocol

All experimental procedures involving animals were conducted in accordance with internationally accepted guidelines for the care and use of laboratory animals and complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The experimental protocol was approved by the Ahi Evran University Animal Experiments Local Ethics Committee, Kırşehir, Turkey (Decision No. 2, Meeting No. 12, Date: 12 June 2025). Every effort was made to minimize animal suffering and to reduce the number of animals used throughout the study. The details of all the reagents and the equipment used are listed in the Table of Materials.

Preparation of chemicals

Nω-nitro-L-arginine methyl ester (L-NAME), indomethacin, iohexol, montelukast sodium, and NAC were used in this study. A 10 mg/mL L-NAME solution was prepared in sterile 0.9% normal saline using 1 g of L-NAME per 100 mL. The solution was freshly prepared on the day of administration and used without storage beyond that day. For induction of the experimental CI-AKI model, L-NAME was administered intravenously (IV) at 10 mg/kg. In a rat weighing approximately 220 g, the calculated amount was approximately 2.2 mg, corresponding to an administration volume of approximately 0.22 mL. Indomethacin was prepared in 5% sodium bicarbonate solution at a concentration of 10 mg/mL by dissolving 100 mg in a final volume of 10 mL. The preparation was vortexed until visually homogeneous. The final pH of the prepared indomethacin solution was not measured during the experiment and therefore could not be retrospectively determined. A specific sterility or filtration procedure for the prepared indomethacin solution was not documented in the experimental records and, therefore, could not be reported retrospectively. No formal stability interval was established or evaluated; the solution was freshly prepared and used on the same day. To induce the experimental CI-AKI model, indomethacin was administered intravenously at 10 mg/kg. For a rat weighing approximately 220 g, the calculated amount was approximately 2.2 mg, corresponding to an administration volume of approximately 0.22 mL.

The commercial iohexol formulation, containing 647 mg iohexol/mL and an equivalent iodine concentration of 300 mg/mL, was administered without additional dilution. The exact temperature of iohexol at the time of injection was not recorded. A formulation-specific osmolality value could not be reliably verified from the available experimental records and is therefore not reported. For induction of the experimental CI-AKI model, iohexol was administered intravenously at 3 g iodine/kg. In a rat weighing approximately 220 g, this dose provided approximately 0.66 g of iodine and corresponded to an injection volume of approximately 2.2 mL.

A montelukast sodium suspension was prepared in 0.5% carboxymethyl cellulose (CMC). It was freshly prepared on each administration day and vortexed immediately before dosing to obtain an even distribution of suspended particles. The montelukast dose required for each animal was calculated separately from the body weight recorded at study initiation using the following formula: required dose (mg) = body weight (kg) × 10 mg/kg. For an animal weighing approximately 220 g, the calculated dose was approximately 2.2 mg and was delivered by oral gavage. The suspension was visually inspected before each administration, and any preparation showing sedimentation was vortexed again before use.

NAC was administered to the treatment groups from its commercial formulation without further dilution at an intraperitoneal dose of 100 mg/kg. For each animal, the required dose was calculated individually from the body weight recorded at study initiation. In an animal weighing approximately 220 g, this corresponded to approximately 22 mg of NAC and an administration volume of approximately 0.15 mL. Before dosing, the solution was examined visually, and only preparations that were clear and free of visible particles were used.

Each preparation underwent visual inspection before administration. The L-NAME, indomethacin, iohexol, and NAC solutions were checked for discoloration and visible particles. Immediately before administration, the montelukast suspension was vortexed and examined visually to confirm uniformity. Any preparation that failed to satisfy these acceptance criteria was not used. Laboratory coats, protective gloves, and eye protection were worn during the preparation and administration of all chemicals. All procedures complied with institutional laboratory safety and biosafety requirements.

Animal selection, housing, experimental groups, and randomization

Thirty adult male Wistar albino rats (8–12 weeks old; body weight, 200–220 g) were obtained from the Experimental Animal Research Laboratory of Ahi Evran University, Kırşehir, Türkiye. Male rats were selected to maintain methodological consistency with previously published experimental CI-AKI models and to avoid introducing estrous-cycle stage as an additional biological variable. Sex was not evaluated as an experimental variable in the present study. The animal facility held a breeding/production license no. 184, and the rats were bred and maintained there under standard laboratory conditions. At enrollment, the animals had no documented specific pathogen-free (SPF) status or other formally assigned microbiological health classification. Each rat was housed separately in a polycarbonate cage measuring 40 cm × 25 cm × 20 cm, with coarsely chopped pine shavings used as bedding. Room conditions were maintained at 24 °C ± 2 °C and 35% ± 10% relative humidity by an automated environmental control and ventilation system. A 12 h light/12 h dark schedule was applied, with illumination from 07:00 to 19:00. No dedicated environmental enrichment was supplied during the 7-day acclimatization phase or the subsequent experimental period. Standard pelleted rodent feed and tap water were available ad libitum, apart from the defined 16 h period of water deprivation preceding CI-AKI induction. The feed composition was 24% crude protein, 3.94% crude cellulose, 5.08% crude fat, 8.8% crude ash, 1.44% lysine, 0.61% methionine, 1.14% calcium, 0.89% phosphorus, and 0.28% sodium. All animals were acclimatized for 7-days before the experimental procedures began. Daily observations were conducted during both the acclimatization and experimental periods to assess general condition, food and water consumption, mobility, posture, coat appearance, and any signs of distress. To minimize selection bias, the animals were allocated to the five experimental groups using a computer-generated random allocation sequence with six animals assigned to each group. The sample size was determined by an a priori power analysis using G*Power version 3.1.9.6.

The sample size was determined by an a priori power analysis for a fixed-effects one-way analysis of variance involving five experimental groups. Assuming an effect size (f) of 0.65, a type I error rate (α) of 0.05, and a statistical power of 80%, the required total sample size was calculated as 30 animals, corresponding to six animals per group. The effect size (f = 0.65) was assumed a priori to represent a large overall between-group effect for the five-group comparison. The calculation was intended to determine the sample size required to detect such an overall effect and was not specifically designed to detect smaller pairwise differences between the active treatment groups. The individual animal was considered the experimental unit for all analyses.

Except for routine husbandry, daily health monitoring, and terminal procedures, animals in the Control group did not undergo protocol-related handling or restraint, water deprivation, intravenous or intraperitoneal injections, oral gavage, or vehicle administration. The CI-AKI group comprised animals in which the CI-AKI model was established on day 8 and that received no treatment. The CI-AKI + Montelukast group comprised animals in which the CI-AKI model was established and that were subsequently treated with montelukast. The CI-AKI + NAC group comprised animals in which the CI-AKI model was established and that were subsequently treated with NAC. The CI-AKI + Montelukast + NAC group comprised animals in which the CI-AKI model was established and that were subsequently treated with a combination of montelukast and NAC. Neither the Control group nor the untreated CI-AKI group underwent treatment-matched sham procedures. Specifically, no sham handling, sham oral gavage, sham treatment injections, or treatment-matched vehicle administrations were performed in these groups. The vehicles used for the preparation of L-NAME, indomethacin, and montelukast were 0.9% normal saline, 5% sodium bicarbonate solution, and 0.5% CMC, respectively, as described in the preparation of chemicals. NAC was administered from its commercial formulation without further dilution, and no separate vehicle was prepared for NAC. On day 8, all animals except those in the Control group underwent 16 h of water deprivation, followed by sequential intravenous administration of L-NAME (10 mg/kg), indomethacin (10 mg/kg), and iohexol (3 g iodine/kg) to induce contrast-induced acute kidney injury. In the designated treatment groups, montelukast (10 mg/kg, oral gavage) and/or NAC (100 mg/kg, intraperitoneal injection) were first administered 1 h after completion of iohexol administration on day 8, corresponding to approximately 1.5 h after L-NAME administration, and were subsequently administered once daily at the same time on days 9 and 10.

Drug administration protocol

CI-AKI was established according to a published experimental protocol consisting of 16 h of water deprivation followed by sequential intravenous administration of L-NAME, indomethacin, and iohexol^23,24,25. L-NAME and indomethacin were each given at 10 mg/kg, whereas iohexol was administered at 3 g iodine/kg. The treatment doses were 10 mg/kg for montelukast sodium and 100 mg/kg for NAC. Selection of the model-induction and treatment doses was guided by previously published experimental studies^22,26,27. No preliminary dose-ranging or dose-optimization study was performed. The NAC and montelukast doses were selected independently based on previously published experimental studies of the individual agents and were not derived from a combination-specific dose-finding study. The selected doses were not intended to represent pharmacologically equipotent exposures, and no dose-response or formal drug-interaction analysis was performed. No prespecified numerical or laboratory threshold was used to define successful model induction at the individual-animal level. At the study endpoint, successful establishment of renal injury was supported by the characteristic histopathological alterations observed in the untreated CI-AKI group compared with the Control group. On day 8, all animals except those in the Control group underwent a single 16 h period of water deprivation immediately before induction of contrast-induced acute kidney injury. Water deprivation was not repeated during the remainder of the experimental period. Standard pelleted chow remained available ad libitum throughout the water-deprivation period. Upon completion of the single 16 h water-deprivation period, access to tap water was restored simultaneously and ad libitum in all water-deprived experimental groups, before administration of the first model-inducing agent, L-NAME, and was maintained for the remainder of the experimental period. The timing and conditions of water restoration were identical across all water-deprived groups. Water intake after restoration was not restricted to a predefined volume and was not quantitatively measured. Paired body weights immediately before and after the 16 h water-deprivation period were not measured; therefore, dehydration-associated percentage body weight loss could not be calculated. Urine output was not quantitatively monitored. Following restoration of water access, induction of CI-AKI was initiated. All sequential intravenous administrations of L-NAME, indomethacin, and iohexol were performed through a lateral tail vein using a 26 G needle attached to a sterile 5 mL syringe. Before intravenous administration, the tail was warmed in water for several minutes to facilitate visualization and dilation of the lateral tail veins. Following each intravenous administration, the vein was flushed with 0.5 mL of sterile 0.9% normal saline, in addition to the calculated administration volume of the respective model-inducing agent. The injection site was visually monitored throughout administration and flushing for swelling, leakage, or evidence of extravasation. L-NAME (10 mg/kg) was administered first. Fifteen minutes after L-NAME administration, indomethacin (10 mg/kg) was administered intravenously through the same lateral tail vein. Fifteen minutes after indomethacin administration, iohexol was administered intravenously through the same lateral tail vein at a dose of 3 g iodine/kg. For each animal, the iohexol dose and corresponding injection volume were calculated individually based on the body weight recorded at study initiation and the commercial iodine concentration of 300 mg iodine/mL. This corresponded to an administration volume of 10 mL/kg and, therefore, approximately 2.0–2.2 mL for rats weighing 200–220 g. No separate protocol-defined maximum permitted intravenous volume was prespecified; the largest single-agent intravenous administration used in this study was iohexol at 10 mL/kg. Iohexol was administered slowly over approximately 1–2 min. Successful intravenous administration was verified by the absence of visible swelling, leakage, or extravasation at the injection site. No partial extravasation was observed, and no injection required reattempt or repeat dosing because of suspected extravasation. One hour after completion of iohexol administration, the designated treatments were first administered to the treatment groups. Because iohexol administration was initiated 30 min after L-NAME administration and completed over approximately 1–2 min, treatment initiation occurred approximately 91–92 min (about 1.5 h) after L-NAME administration. Montelukast sodium (10 mg/kg) was administered by oral gavage, whereas NAC (100 mg/kg) was administered intraperitoneally.

Oral gavage was performed using a 22 G stainless-steel feeding cannula. Each animal was maintained in an upright position, and the feeding cannula was gently advanced through the esophagus into the stomach. If excessive resistance was encountered, the cannula was withdrawn and repositioned before administration. The animal was observed for regurgitation during and immediately after gavage. No regurgitation or other evidence of incomplete oral dosing was observed during or immediately after gavage in any animal, and no animal was excluded on the basis of suspected unsuccessful oral administration. Intraperitoneal injections were administered into the lower right quadrant of the abdomen. The needle was inserted at an angle of approximately 30°–45°, and aspiration was performed before administration to check for inadvertent vascular entry. The injection site was visually monitored during and immediately after administration for leakage, bleeding, or other visible complications. The designated treatments were administered once daily at the same time on days 8, 9, and 10. Accordingly, animals in the CI-AKI + Montelukast group received montelukast once daily for three consecutive days, animals in the CI-AKI + NAC group received NAC once daily for three consecutive days, and animals in the CI-AKI + Montelukast + NAC group received both montelukast and NAC once daily according to the same 3-day treatment schedule. All model-induction and treatment administrations were performed by the same investigator. Body weight was measured once for each animal at study initiation and was not remeasured during the experimental period. For each animal, the dose and corresponding administration volume of all model-inducing agents and study treatments were calculated individually using the body weight recorded at study initiation. All intravenous, intraperitoneal, and oral gavage procedures were performed using the same standardized technique to minimize administration-related variability.

Euthanasia procedure, humane endpoints, and sample collection

Animals were observed once daily throughout the experiment for changes in general condition, food and water consumption, mobility, posture, coat appearance, and evidence of distress. The prospectively defined humane endpoints comprised marked lethargy, inability to access or consume available food or water, abnormal posture, reduced mobility, persistent piloerection, and any other overt manifestation of distress. Animals meeting any of these criteria were to undergo immediate euthanasia; however, no animal fulfilled the humane endpoint criteria during the study. On day 11, 24 h after the final treatment, deep anesthesia was induced by intraperitoneal administration of ketamine (80 mg/kg) together with xylazine (10 mg/kg). The absence of the pedal withdrawal response was used to verify an adequate anesthetic plane. Once sufficient anesthetic depth had been confirmed, approximately 3–5 mL of blood was obtained from each animal by intracardiac puncture. Euthanasia was then completed by exsanguination while the animal remained under deep anesthesia. The absence of both cardiac activity and spontaneous respiration was used to confirm death.

Blood collected for serum biochemical measurements was placed in anticoagulant-free tubes and left until clotting was complete. The tubes were then centrifuged at 3,000 × g for 10–15 min at 4 °C, after which the serum fraction was separated and stored at −80 °C until analysis. Samples intended for glutathione measurement were collected separately in tubes containing ethylenediaminetetraacetic acid (EDTA). Because these specimens were anticoagulated, they were processed without a clotting stage in accordance with the corresponding glutathione assay procedure. Serum separation was considered acceptable when the recovered serum was clear and showed no visible evidence of hemolysis.

Once death had been confirmed, a median laparotomy was carried out and both kidneys were carefully removed from each animal. Surrounding adipose and connective tissues were cleared from the specimens. The right kidney was reserved solely for histopathological examination, whereas the left kidney was assigned to biochemical analysis. Mixed tissue from the left kidney was analyzed without separately dissecting the renal cortex and medulla. The two kidneys were handled independently, and specimens from different animals remained separate throughout processing without pooling. The entire right kidney was placed in 10% neutral buffered formaldehyde and processed for histopathological assessment. Sections encompassing the renal cortex and outer medulla were prepared and examined according to the procedures described in the histopathological processing and scoring. Each left kidney was labeled individually and maintained at -80 °C until homogenization and biochemical testing. After excision, gross tissue integrity was checked, and an adequate amount of tissue was retained for the intended histopathological and biochemical assessments.

The investigator responsible for model induction and treatment administration could not be blinded because the experimental groups underwent different procedures. However, group identities were concealed from the investigators conducting the biochemical and statistical analyses and from the pathologist performing the histopathological assessments. All enrolled animals were retained in the study and included in the subsequent analyses. Biological samples, animal tissues, formaldehyde-based fixatives, and other chemical waste materials were handled with appropriate personal protective equipment. Disposal of all biological and chemical waste was carried out in compliance with institutional biosafety and hazardous-waste management requirements.

Biochemical analyses

Biochemical measurements were conducted using serum, EDTA-anticoagulated blood, and left-kidney tissue homogenates collected from each animal. The procedures are presented below according to the biological matrix used for each parameter. Serum was analyzed for urea, blood urea nitrogen (BUN), creatinine, superoxide dismutase (SOD), and malondialdehyde (MDA). Urea, BUN, and creatinine served as renal function indices, whereas SOD activity and MDA concentration were evaluated as oxidative stress parameters. Serum urea and BUN values were obtained using the same enzymatic urease–glutamate dehydrogenase assay. In this method, urea was hydrolyzed by urease to produce ammonia, which subsequently reacted with 2-oxoglutarate and reduced nicotinamide adenine dinucleotide (NADH) in the presence of glutamate dehydrogenase. The analytical method used was an enzymatic urease–glutamate dehydrogenase kinetic assay. The decrease in absorbance resulting from NADH consumption was measured kinetically using a spectrophotometer. Serum creatinine concentration was measured colorimetrically using an automated biochemistry analyzer. Serum SOD activity was measured colorimetrically using a commercially available assay kit on the same automated biochemistry analyzer. The SOD assay was based on the generation of superoxide radicals by the xanthine–xanthine oxidase system and their reaction with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride to form a colored formazan product. The endpoint absorbance was measured at 505 nm, and SOD activity was expressed as U/mL. Serum MDA concentration was determined colorimetrically using the thiobarbituric acid reaction. Serum samples were mixed with cold trichloroacetic acid to precipitate proteins, and the resulting precipitate was separated by centrifugation. This centrifugation step was performed at 7,500 × g for 5 min at 4 °C. The supernatant was reacted with thiobarbituric acid at 90 °C for 60 min, cooled, and measured at 532 nm. MDA concentrations were calculated from the corresponding calibration curve and expressed as nmol/mL. Glutathione (GSH) analysis was performed using EDTA-anticoagulated blood. Proteins were precipitated with trichloroacetic acid, and the resulting deproteinized samples were analyzed colorimetrically by a modified Ellman method. The samples were combined with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in 500 mM Tris buffer at pH 8.2. Reaction of the thiol groups of GSH with DTNB generated a colored product, and absorbance was recorded at 412 nm. Catalase (CAT) activity was determined using a two-step colorimetric procedure. Samples were initially incubated with a known amount of hydrogen peroxide, which catalase converted to water and molecular oxygen. After the enzymatic reaction was terminated, residual hydrogen peroxide was measured using a chromogenic reagent. Absorbance was recorded at 405 nm, and CAT activity was reported as U/mL. For renal tissue measurements, the left kidney from each animal was used as mixed tissue without separating the cortex from the medulla. As stated in the procedure, the right kidney was retained solely for histopathological assessment. Samples belonging to individual animals were processed independently and were not pooled at any stage. Each left-kidney specimen was homogenized on ice in 50 mM phosphate buffer at pH 7.4 using a tissue-to-buffer ratio of 1:10 (w/v). One animal-specific renal tissue homogenate was generated for each rat. Separate aliquots of this homogenate were used for the different renal tissue measurements, ensuring that all parameters were derived from the same individual animal. The resulting renal tissue homogenates were centrifuged at 14,000 × g for 10 min at 4 °C. Following centrifugation, the supernatant obtained from each animal was collected separately and divided into aliquots for the planned renal tissue analyses. Tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), cystatin C, and KIM-1 concentrations in the renal tissue supernatants were measured using rat-specific enzyme-linked immunosorbent assay (ELISA) kits. The assays were performed according to the manufacturer’s protocols. The incubation conditions were identical for the TNF-α, IL-1β, IL-6, cystatin C, and KIM-1 ELISAs. For each assay, the samples and ELISA reagents were incubated for 1 h at 37 °C; after five washing cycles, substrate solutions A and B were added and incubated for 10 min at 37 °C. Briefly, standards and renal tissue supernatants were added to the antibody-precoated wells, followed by the corresponding biotinylated detection antibody and streptavidin–horseradish peroxidase conjugate. After the specified incubation steps, unbound components were removed using an automated microplate washer. The substrate solution was subsequently added, and the color reaction was terminated using the acidic stop solution. Absorbance was measured at 450 nm using a microplate reader, and analyte concentrations were calculated from the corresponding standard curves. Renal tissue ELISA measurements were performed in duplicate. For each analyte, the mean of the two technical replicate wells was calculated to obtain a single animal-level value. Technical replicate wells and separate aliquots obtained from the same animal-specific homogenate were not considered independent observations. All biochemical measurements were performed under standardized assay-specific conditions. Serum, EDTA-anticoagulated blood, and renal tissue samples were processed separately according to the requirements of the corresponding analytical methods. Investigators performing the biochemical analyses were blinded to group allocation. The individual animal, rather than each serum sample, blood sample, kidney specimen, homogenate aliquot, or technical replicate well, was considered the experimental unit. Accordingly, six independent animal-level observations were included in the statistical analysis for each experimental group.

Histopathological processing and scoring

Right-kidney specimens were prepared for microscopic evaluation by routine histological processing. The whole right kidney from each animal was immersed in 10% neutral buffered formaldehyde for 72 h at room temperature. After fixation, tissue dehydration was performed sequentially in 50%, 70%, 80%, 96%, and 100% ethanol, with each concentration applied for 1 h. The specimens were then exposed to two xylene baths for 30 min per bath before paraffin embedding. Using a rotary microtome, 5 µm sections that included both the renal cortex and outer medulla were obtained from the paraffin blocks.

For hematoxylin and eosin (HE) staining, the sections were deparaffinized and rehydrated, exposed to hematoxylin for 5 min, and washed under running tap water for an additional 5 min. Acid alcohol was used for differentiation, after which the sections were blued in ammoniated water for 1 min and counterstained with eosin for 2 min. The stained sections were subsequently dehydrated in ascending alcohol concentrations, cleared with xylene, and mounted permanently under coverslips.

Sections designated for Masson’s trichrome staining were likewise deparaffinized and rehydrated. Mordant treatment was performed for 60 min at 56 °C, followed by rinsing under running tap water for 10 min. The sections were then treated sequentially with Weigert’s iron hematoxylin for 10 min, Biebrich scarlet–acid fuchsin solution for 15 min, phosphotungstic acid solution for 10 min, and aniline blue solution for 10 min. After immersion in 1% acetic acid for 5 min, the sections were dehydrated, cleared in xylene, and mounted with coverslips.

Before microscopic assessment, the adequacy of staining was verified separately for each method. HE-stained sections were considered suitable when nuclear details and cytoplasmic contrast were clearly discernible, whereas Masson’s trichrome-stained sections were accepted when connective tissue structures could be distinctly visualized. For each animal, three HE-stained sections and three Masson’s trichrome-stained sections were examined. Five randomly selected microscopic fields were evaluated in each section using a 40× objective. Accordingly, 15 microscopic fields per staining method were examined for each animal. All microscopic examinations were performed using the same objective and standardized evaluation conditions across all animals and experimental groups. Masson’s trichrome-stained sections were evaluated qualitatively for interstitial collagen distribution and deposition, extracellular matrix accumulation, and tubulointerstitial architecture. These sections were not used to generate the semi-quantitative 0–3 histopathological injury scores. Representative HE- and Masson’s trichrome-stained photomicrographs were acquired using a 40× objective, and a 100 µm scale bar was included in each representative image. These photomicrographs were obtained for illustrative purposes and were not treated as independent observations in the histopathological scoring or statistical analyses. All histopathological assessments were conducted by one experienced pathologist who remained blinded to the experimental group identities. Using a single blinded examiner was intended to limit assessment bias and variation attributable to differences between observers. Tubular necrosis, tubular dilatation, cellular degeneration, and vascular congestion were scored semi-quantitatively on HE-stained renal sections according to a system adapted from previously published renal injury models^28,29. For each parameter, scores ranged from 0 to 3, corresponding to absent, mild, moderate, and severe histopathological damage, respectively. For each animal, the findings from the five microscopic fields examined in each of the three HE-stained sections, corresponding to 15 fields in total, were considered collectively to assign a single 0–3 score for each histopathological parameter. Thus, each animal contributed one score for tubular necrosis, tubular dilatation, cellular degeneration, and vascular congestion. Histological sections and microscopic fields were treated as within-animal subsamples and were not considered independent observations. Within each section, microscopic fields were randomly selected from comparable renal cortical and outer medullary regions to standardize anatomical sampling across animals and experimental groups. The individual animal, rather than each kidney, histological section, or microscopic field, was considered the experimental unit. Accordingly, six independent animal-level observations were included in the statistical analysis for each experimental group. The values presented in Table 3 represent the group means calculated from the individual animal-level scores of the six animals in each experimental group.

Statistical analysis

Statistical analyses were performed using statistical analysis software. The individual animal was considered the experimental unit for all statistical analyses. Each experimental group comprised six independent animals (n = 6 per group). Technical replicates, assay wells, tissue sections, and microscopic fields were not treated as independent observations and were not used to increase the statistical sample size. Continuous variables are presented as mean ± standard deviation (SD). Normality was evaluated separately within each experimental group using the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene’s test. Given the fully balanced experimental design with equal sample sizes across groups, a one-way analysis of variance (ANOVA) was used to compare groups. Pairwise comparisons were performed using Duncan’s post hoc multiple comparison test. Potential outliers were evaluated by graphical examination of boxplots and inspection of individual observations. No observation was considered to represent a measurement error or an invalid experimental result, and no data point was excluded on statistical grounds. A p-value < 0.05 was considered statistically significant.

Results

Serum kidney function markers (urea, blood urea nitrogen, and creatinine)

The experimental design, animal grouping, treatment schedule, and sample collection timeline of the present study are summarized in Figure 1. Statistically significant differences were observed among the groups for serum urea, BUN, and creatinine levels (p = 0.003, p = 0.003, and p = 0.002, respectively). The mean serum urea l...

Discussion

This experimental CI-AKI study evaluated post-contrast treatment with montelukast, NAC, or their combination across renal function, oxidative stress parameters, inflammatory responses, renal injury biomarkers, and histopathological outcomes. Contrast exposure produced substantial increases in serum urea, BUN, and creatinine and was accompanied by adverse changes in oxidative stress parameters and inflammatory biomarkers. Increased KIM-1 and cystatin C levels provided additional evidence of renal injury.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Auto Strip Washer	Bio-Tek Instruments, USA	ELx50	Automated ELISA plate washing.
Microplate Reader	Bio-Tek Instruments, USA	ELx800	ELISA absorbance measurements. RRID:SCR_028651
10% Neutral Buffered Formalin	Merck	Not recorded	Tissue fixation for histopathology.
Benchtop Centrifuge	Nüve	NF048	Sample preparation and centrifugation.
Carboxymethylcellulose (CMC)	Sigma-Aldrich, USA	C5678	Vehicle used for montelukast suspension.
CAT Kit Universal	Otto Scientific	Otto3051	Colorimetric measurement of CAT activity in serum using a Mindray BS-400 chemistry analyzer.
Digital Homogenizer	Daihan Scientific	HG-15D	Tissue homogenization.
Eosin Y (Yellowish)	Merck	1.15935.0025	HE staining.
Harris Hematoxylin Solution	BSLAB	BS-002	HE staining.
Indomethacin	Sigma-Aldrich, USA	I7378	Used for induction of CI-AKI.
Iohexol (Omnipaque 300)	GE HealthCare	Not recorded	Contrast agent used to induce CI-AKI.
Ketamine hydrochloride (Ketalar)	Pfizer, USA	Not recorded	General anesthesia before sample collection.
Light Microscope (Nikon Eclipse)	Nikon	Ni-U	Histopathological examination and imaging. RRID:SCR_024835
L-NAME (Nω-nitro-L-arginine methyl ester)	Sigma-Aldrich, USA	N5751	Used for induction of CI-AKI.
Masson's Trichrome Staining Kit	MOSLAB, Türkiye	BKMTO001	Masson trichrome staining.
Mindray Chemistry Analyzer	Mindray	BS-400	Biochemical analyses.
Montelukast sodium	Sigma-Aldrich, USA	SML0101	Administered orally by gavage as treatment.
N-acetylcysteine (ASIST 300 mg/2 mL)	MAC laç, Türkiye	Not recorded	Administered intraperitoneally as treatment.
Rat Cystatin C ELISA Kit	BT Lab, China	E0145Ra	ELISA measurement of cystatin C in renal tissue supernatants.
Rat IL-1β ELISA Kit	BT Lab, China	E0119Ra	ELISA measurement of IL-1β in renal tissue supernatants.
Rat IL-6 ELISA Kit	BT Lab, China	E0135Ra	ELISA measurement of IL-6 in renal tissue supernatants.
Rat KIM-1 ELISA Kit	BT Lab, China	E0549Ra	ELISA measurement of KIM-1 in renal tissue supernatants.
Rat TNF-α ELISA Kit	BT Lab, China	E0764Ra	ELISA measurement of TNF-α in renal tissue supernatants.
Stainless Steel Feeding Needle (22 G Oral Gavage Cannula)	Instech Laboratories, USA	Not recorded	Oral gavage administration.
Standard pelleted rodent chow (Optima)	Optima, Türkiye	Not recorded	Lot no. 58931. Nutritional composition: 24% crude protein, 3.94% crude cellulose, 5.08% crude fat, 8.8% crude ash, 1.44% lysine, 0.61% methionine, 1.14% calcium, 0.89% phosphorus, and 0.28% sodium.
Universal Creatinine Kit	Otto Scientific	OttoBC139	Colorimetric measurement of creatinine in serum.
Universal GSH Kit	Otto Scientific	Otto3053	Colorimetric measurement of GSH in EDTA-anticoagulated blood using a modified Ellman method.
Universal MDA Kit	Otto Scientific	Otto1001	Colorimetric measurement of MDA in serum using an ELx800 microplate reader.
Universal SOD Kit	Otto Scientific	Otto3047	Colorimetric measurement of SOD activity in serum using a Mindray BS-400 chemistry analyzer.
Universal Urea Kit	Otto Scientific	OttoBC157	Colorimetric measurement of urea and BUN in serum.
Xylazine hydrochloride (Rompun)	Bayer, Germany	Not recorded	General anesthesia before sample collection.