Protective Effect of Wogonin Against Colistin-Induced Nephrotoxicity in Wistar Rats: A Controlled Experimental Study

Protective Effect of Wogonin Against Colistin-Induced Nephrotoxicity in Wistar Rats: A Controlled Experimental Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Protective Effect of Wogonin Against Colistin-Induced Nephrotoxicity in Wistar Rats: A Controlled Experimental Study Ahmet Salih Tüzen, Birzat Emre Gölboyu, Mehmet Ali Coşar, Mümin Alper Erdoğan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7124382/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Colistin remains a critical antibiotic for treating multidrug-resistant Gram-negative infections, yet its clinical use is severely limited by its high nephrotoxicity profile. There is an urgent need for nephroprotective agents that can mitigate colistin-induced nephrotoxicity (CIN) without compromising antimicrobial efficacy. Wogonin, a flavonoid compound with known antioxidant and anti-inflammatory properties, has shown promise in various models of renal injury, though its potential role in CIN has not been previously evaluated. This study aimed to assess the nephroprotective efficacy of wogonin in a well-characterized Wistar rat model of CIN, using a combination of biochemical, molecular, and histopathological parameters. Thirty adult female Wistar rats were randomly assigned into three groups: control, colistin + tap water, and colistin + wogonin (50 mg/kg/day, oral gavage for 10 days). CIN was induced using a single intraperitoneal dose of colistin (20 mg/kg). Plasma levels of blood urea nitrogen (BUN), creatinine, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were measured, along with renal tissue levels of SIRT-1 and Nrf2. Histopathological evaluation was performed using a semi-quantitative scoring system. Colistin administration resulted in significant renal dysfunction and tubular injury, as evidenced by elevated biochemical markers and histological damage. Wogonin treatment significantly attenuated elevations in BUN, creatinine, KIM-1, NGAL, MDA, IL-6, and TNF-α, while restoring sirtuin-1 (SIRT-1) and nuclear factor erythroid 2–related factor 2 (Nrf2) levels. Histopathological analysis revealed reduced tubular necrosis, luminal debris, and dilatation in the wogonin group. Wogonin demonstrated significant nephroprotective effects in a rat model of CIN by reducing oxidative stress, suppressing pro-inflammatory cytokine expression, and mitigating tubular epithelial injury. Biological sciences/Biochemistry Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Health sciences/Nephrology Colistin colistin-induced nephrotoxicity inflammation nephroprotection oxidative stress Scutellaria baicalensis wogonin Figures Figure 1 Figure 2 Introduction Polymyxins, particularly colistin, have re-emerged as last-resort antibiotics for treating multidrug-resistant Gram-negative bacterial infections, especially in critically ill patients. Despite its clinical significance, the therapeutic utility of colistin is greatly restricted due to its nephrotoxic potential, which affects approximately 20–50% of treated patients in a dose- and duration-dependent manner 1–4 . The nephrotoxicity primarily manifests as acute kidney injury (AKI), involving oxidative stress, inflammation, and tubular epithelial damage 3,5 . Experimental models have consistently shown that colistin induces oxidative stress, mitochondrial dysfunction, lipid peroxidation, and apoptosis, leading to characteristic histological alterations such as tubular epithelial necrosis, interstitial inflammation, and loss of brush border integrity. These pathophysiological mechanisms, confirmed by elevations in plasma creatinine, blood urea nitrogen (BUN), and lipid peroxidation markers such as malondialdehyde (MDA) levels, underline its potent nephrotoxic profile 6–8 . Even in clinical scenarios where colistin remains indispensable, its use is often approached with caution due to the high likelihood of renal complications; particularly in vulnerable, critically ill populations with limited renal reserve 1 . In this context, there remains a critical and urgent need for effective adjunctive therapies that can protect the kidneys from CIN without compromising its potent antimicrobial activity 1,2,4 . Among various mechanistic contributors to CIN, pro-inflammatory mediators such as interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) play important roles in propagating renal inflammation and injury 9,10 . Within this pathophysiological framework, natural compounds possessing antioxidant and anti-inflammatory properties have garnered increasing interest as promising adjunctive therapies for mitigating nephrotoxic insults. Wogonin (5,7-dihydroxy-8-methoxy flavone), a natural flavonoid derived from the roots of Scutellaria baicalensis , has attracted growing interest due to its diverse pharmacological properties, particularly its antioxidant, anti-inflammatory, and anti-fibrotic effects 11 . These attributes have positioned it as a promising candidate in experimental models of nephrotoxicity. In various preclinical studies, wogonin has been shown to exert nephroprotective effects by modulating critical molecular pathways, including the inhibition of NF-κB, Transforming Growth Factor-beta (TGF-β) / Mothers against decapentaplegic homolog 3 (Smad3), and Phosphatidylinositol 3-Kinase (PI3K) / Protein Kinase B (Akt) signaling, while enhancing antioxidant defenses through activation of the Nuclear factor erythroid 2–related factor 2 (Nrf2) / Heme oxygenase-1 (HO-1) pathway. Additionally, it reduces the expression of key pro-inflammatory cytokines such as TNF-α and Interleukin-6 (IL-6), thereby attenuating renal inflammation, oxidative stress, and fibrotic remodeling 9–13 . These mechanisms have been validated in several kidney injury models, including diabetic nephropathy, cisplatin-induced AKI, and unilateral ureteral obstruction, where wogonin consistently demonstrated protective effects on renal structure and function 9,10,12–15 . Despite its broad-spectrum nephroprotective actions in various experimental models, no published studies have yet systematically evaluated the potential protective effects of wogonin in CIN. Given that CIN is mediated by distinct injury mechanisms, such as mitochondrial dysfunction, lipid peroxidation, and pro-inflammatory signaling, translating wogonin’s known nephroprotective mechanisms to this specific context is of high clinical relevance 5,8 . The lack of histopathological and biochemical studies investigating the effects of wogonin in CIN models demonstrate a significant gap in the current literature. To address this, we designed a randomized controlled experimental study using adult female Wistar rats to evaluate the nephroprotective efficacy of wogonin against CIN. Our primary hypothesis was that wogonin, through its modulation of oxidative stress and inflammatory pathways, would mitigate renal injury and preserve renal function and morphology. To investigate this, the present study primarily focused on the histopathological assessment of renal tissue using detailed semiquantitative scoring. As secondary objectives, we evaluated a comprehensive panel of renal biomarkers in both serum and tissue samples, including functional markers: BUN, creatinine; early injury biomarkers: Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL); inflammatory mediators: MDA, IL-6, and TNF-α, and oxidative stress-related regulators: Sirtuin-1 (SIRT-1) and Nrf2. These parameters were selected to explore the mechanistic basis of wogonin’s potential nephroprotective activity. Accordingly, this study aims to investigate the nephroprotective efficacy of wogonin as a adjunctive therapeutic agent against CIN using a randomized in vivo rat model. Materials and Methods Ethical Considerations All procedures involving animals were reviewed and approved by the Institutional Animal Care and Ethical Committee of the Science University, Istanbul (Approval Number: 2925042821), and conducted in full compliance with the internationally recognized ARRIVE 2.0 guidelines 16 . All methods were performed in accordance with the relevant guidelines and regulations. The study was meticulously designed to ensure the ethical treatment of animals, with particular emphasis on the principles of replacement, reduction, and refinement (the 3Rs) 17 . Every effort was made to minimize pain, distress, and suffering throughout the experimental protocol. All animals were closely monitored during the study period to ensure humane care and early intervention in the case of any adverse effects. Study Design and Animals This controlled in vivo experimental study was conducted using 30 adult female Wistar rats ( Rattus norvegicus ), each weighing between 200–210 grams and aged 8-10 weeks, obtained from the Central Animal Facility of Science University, Istanbul, Turkey. Upon procurement, all animals underwent a one-week acclimatization period to minimize environmental stress and allow for physiological and behavioral stabilization. Animals were housed in standard ventilated polycarbonate cages, five rats per cage, under controlled environmental conditions; ambient temperature of 22 ± 2 °C, relative humidity maintained at 50-60%, and a 12-hour light/dark cycle. Environmental enrichment strategies, including nesting materials and shelter objects, were uniformly applied to promote natural behaviors and improve welfare. Rats were provided ad libitum access to standardized pellet chow and filtered tap water for the entire duration of the study. Each animal was examined daily for clinical signs of illness or distress, including monitoring for changes in body weight, grooming behavior, food and water intake, and overall activity. Humane endpoints were established prior to study initiation and were strictly enforced to prevent unnecessary suffering. Experimental Groups and Treatment Protocol Given the complex pathophysiology of CIN, encompassing oxidative stress, inflammation, apoptosis, and structural renal damage, in vivo models are indispensable. Accordingly, our study employed an animal model to assess pharmacokinetic and pharmacodynamic responses, immunological effects, and renal structural changes following colistin exposure and wogonin treatment. The treatment regimen and experimental protocol were designed to evaluate the therapeutic efficacy of wogonin in attenuating CIN. A total of 30 adult female Wistar rats were enrolled in the study and randomly assigned into three groups (n=10 per group) to minimize selection bias. First group served as the normal control group and received no pharmacological intervention during the study period. The second group (colistin + tap water group) and the third group (colistin + wogonin group) were administered a single intraperitoneal injection of colistin at a dose of 20 mg/kg to induce AKI. Following colistin (Kocak Pharma, Istanbul, Turkey) administration, colistin + tap water group rats received 1 mL/kg/day of tap water via oral gavage for 10 consecutive days, while colistin + wogonin group received wogonin (Adipogen, San Diego, CA, USA) at a dose of 50 mg/kg/day via oral gavage for the same duration. Colistin dosage was selected based on prior studies demonstrating reproducible AKI in rodent models 6,18 . The dosage and administration method for wogonin were selected based on previous preclinical studies demonstrating its nephroprotective effects in rodent models of renal injury 15 . During the treatment period, two animals from colistin + tap water group died, presumably due to severe CIN. These animals were excluded from subsequent biochemical and histological analyses. No mortality was observed in either the control group or the wogonin-treated group. Study Outcomes The primary outcome of this study was the evaluation of renal histopathological alterations using semiquantitative scoring to determine the extent of tubular epithelial damage, including necrosis, tubular dilatation, and luminal debris, in response to colistin and the potential protective effect of wogonin. The secondary outcomes included assessment of renal function through biochemical markers such as BUN and serum creatinine, early tubular injury biomarkers including KIM-1 and NGAL, and systemic and tissue-specific inflammatory and oxidative stress parameters. These included MDA, TNF-α, IL-6, and antioxidant regulators such as SIRT-1 and Nrf2. These outcome measures were selected to comprehensively evaluate the pathophysiological impact of colistin and to determine the nephroprotective efficacy of wogonin at both functional and molecular levels. Sample Collection and Sacrifice At the conclusion of the 10-day treatment period, all surviving animals were anesthetized using a high-dose intraperitoneal injection of ketamine (100 mg/kg) (Vemilac, Istanbul, Turkey) and xylazine (10 mg/kg) (Bioveta, Ivanovice na Hané, Czech Republic) to ensure deep surgical anesthesia prior to tissue and blood collection. Following confirmation of adequate anesthesia, the kidneys were rapidly excised, rinsed with ice-cold saline to remove residual blood, and processed for both biochemical and histopathological examinations. One portion of each kidney was fixed in 10% buffered formalin for histological analysis, while the remaining tissue was snap-frozen and stored at −20 °C for subsequent biochemical assays. Blood samples were obtained via cardiac puncture using a sterile 1 mL syringe and immediately transferred into heparinized collection tubes for biochemical analysis. Subsequently, the animals were sacrificed by blood collection with cardiac puncture, in accordance with the predefined humane endpoints and ethical guidelines. Biochemical, Oxidative Stress Parameters, and Histopathological Evaluation Plasma samples were obtained by centrifugation of collected blood at 3000 rpm for 10 minutes at room temperature. The separated plasma was aliquoted and stored at −20 °C until analysis. Renal function was evaluated by measuring plasma levels of BUN and creatinine using a Beckman-Coulter AU 640 automated analyzer (Beckman-Coulter Inc., CA, USA). To assess systemic inflammation, plasma concentrations of IL-6, TNF-α, as well as the tubular damage biomarkers KIM-1 and NGAL, were quantified using rat-specific enzyme-linked immunosorbent assay (ELISA) kits (Sigma-Aldrich, St. Louis, MO, USA). Oxidative stress was assessed by measuring MDA concentrations, an indicator of lipid peroxidation, using the thiobarbituric acid reactive substances (TBARS) method with MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA). MDA concentrations were expressed in nanomoles (nM), with tetraethoxypropane used for calibration. Trichloroacetic acid and TBARS reagent were added to plasma samples and incubated at 100 °C for 60 minutes. After cooling on ice, the samples were centrifuged at 3000 rpm for 20 minutes, and the absorbance of the supernatant was read at 535 nm using a spectrophotometer. For renal tissue analysis, whole kidneys were homogenized in phosphate-buffered saline (pH=7.4) at a ratio of 1:5 (w/v) using a glass homogenizer. Homogenates were centrifuged at 5000 × g for 15 minutes, and supernatants were collected for biochemical evaluation. Total protein concentration was determined using the Bradford assay, with bovine serum albumin as the reference standard. Tissue levels of SIRT-1 and Nrf2 were measured from homogenates using rat-specific ELISA kits (Sigma-Aldrich, St. Louis, MO, USA). For histopathological evaluation, kidney tissues were fixed in 10% neutral-buffered formalin for 48 hours, embedded in paraffin blocks, and sectioned at 4 μm thickness. Sections were stained with hematoxylin and eosin (H&E) and examined under light microscopy. All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope (Olympus Co., Tokyo, Japan). Morphological assessments were performed using a computerized image analysis system (Image-Pro Express 1.4.5, Media Cybernetics, Rockville, MD, USA) across 10 non-overlapping fields per section at ×20 magnification. The examiner was blinded to the treatment groups to ensure objectivity. A semi-quantitative scoring system was used to evaluate the degree of tubular epithelial necrosis, luminal necrotic debris, tubular dilatation, and interstitial inflammation. The scoring scale was as follows: 0 (0–5%), 1 (6–20%), 2 (21–40%), 3 (41–60%), 4 (61–80%), and 5 (81–100%) 19 . Composite injury scores were computed to reflect the overall histological damage for each rat. Statistical Analysis All statistical analyses were conducted using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). The normality of data distribution was assessed using the Shapiro-Wilk test to determine the appropriate statistical method for each variable. For parametric data, comparisons between groups were performed using Student’s t-test or one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple intergroup comparisons when indicated. For non-parametric data, the Mann–Whitney U test was used to compare differences between groups. All data are expressed as mean ± standard error of the mean (SEM). A p -value < 0.05 was considered statistically significant for all analyses. A priori power analysis was performed using G*Power version 3.1.9.6 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) 20 to determine the minimum number of animals required per group to detect statistically significant differences among experimental groups. The analysis was informed by histopathological evaluation from previous preclinical studies investigating nephroprotective interventions in AKI models, using a Cohen’s effect size of 0.8, a significance level (α) of 0.05, and a statistical power (1−β) of 0.95. Based on these parameters, the estimated sample size for one-way ANOVA with post hoc comparisons was calculated to be at least eight rats per group. To accommodate potential attrition due to CIN, we included 10 animals in each group, accounting for an anticipated dropout rate of approximately 20%. Results The experimental study flow chart and group distribution are outlined in Fig 1. A total of 30 adult female Wistar rats were initially enrolled. Two rats from the colistin + tap water group died during the 10-day treatment period and were excluded from all analyses. No mortality was observed in either the normal control group or the colistin + wogonin group. Histopathological evaluation of renal injury are summarized in Table 1. Histopathological scores for tubular epithelial necrosis, luminal necrotic debris, and tubular dilatation were higher in the colistin + tap water group compared to the control group ( p < 0.001). These scores were lower in the colistin + wogonin group than in the colistin + tap water group ( p < 0.01). However, no significant differences in interstitial inflammation scores were observed across the groups (Table 1). The kidney histopathological sections are shown in Fig 2, including H&E Staining images from all three groups. Table 1. Histopathological Evaluation of Renal Injury Normal control Group (n=10) Colistin + Tap Water Group (n=8) Colistin + Wogonin Group (n=10) Histopathological score Tubular epithelial necrosis 0.1± 0.2 2.5 ± 0.2 * 0.9 ± 0.1 # Luminal necrotic debris 0.1 ± 0.1 2.2 ± 0.1 * 1.1 ± 0.2 # Tubular dilatation 0.2 ± 0.1 1.7 ± 0.3 * 0.8 ± 0.3 # Interstitial inflammation 0.2 ± 0.1 0.3 ± 0.1 0.2 ± 0.2 Results were presented as mean ± SEM. *p<0.001, (different from control group); # p<0.01 (different from Colistin and tap water group). Plasma levels of creatinine and BUN were measured to assess renal function. Both parameters were elevated in the colistin + tap water group relative to the normal control group ( p < 0.01). In the colistin + wogonin group, plasma creatinine and BUN levels were lower than those in the colistin + tap water group ( p < 0.001) (Table 2). Also, early markers of renal injury, the plasma levels of KIM-1 and NGAL were higher in the colistin + tap water group compared to the normal control group ( p < 0.01). These levels were reduced in the colistin + wogonin group relative to the colistin + tap water group ( p < 0.05) (Table 2). A key lipid peroxidation molecule, plasma MDA levels were elevated in the colistin + tap water group compared to controls ( p < 0.001). Lower levels of MDA were observed in the colistin + wogonin group relative to the colistin + tap water group ( p < 0.05). Similarly, the pro-inflammatory cytokines, plasma concentrations of TNF-α and IL-6 were also elevated in the colistin + tap water group (respectively, p < 0.01 and p < 0.001). Both markers were lower in the colistin + wogonin group than in the colistin + tap water group ( p < 0.05) (Table 2). Renal tissue levels of SIRT-1 and Nrf2 were lower in the colistin + tap water group than in the control group (respectively, p < 0.01 and p < 0.001). These levels were higher in the colistin + wogonin group compared to the colistin + tap water group (respectively, p < 0.05 and p < 0.001) (Table 2). Table 2. Plasma Biochemical Parameters and Renal Tissue Biomarkers Normal control Group (n=10) Colistin + Tap Water Group (n=8) Colistin + Wogonin Group (n=10) Renal Functional and Injury Biomarkers in Plasma BUN (mg/dl) 20.2 ± 1.4 41.1 ± 2.5 * 27.3 ± 0.9 ## Creatinine (mg/dl) 0.38 ± 0.09 1.17 ± 0.2 * 0.74 ± 0.1 # KIM-1(pg/ml) 27.2 ± 0.8 201.5 ± 4.6 ** 86.9 ± 3.1 ## NGAL (pg/ml) 45.5 ± 3.1 116.2 ± 5.9 ** 67.6 ± 4.4 ## Oxidative Stress and Inflammatory Parameters Plasma MDA (nM) 38.7 ± 2.4 77.1 ± 4.5 ** 54.8 ± 2.9 # Plasma TNF-alfa (pg/ml) 19.3 ± 1.1 46.2 ± 2.5 * 30.2 ± 1.1 # Plasma IL-6 (ng/ml) 1.16 ± 0.2 98.5 ± 8.4 ** 41.8 ± 5.08 # Renal Tissue Biomarkers Kidney SIRT-1 Level (pg/mg protein) 1.95 ± 0.1 0.86 ± 0.2 * 1.22 ± 0.09 # Kidney Nrf-2 Level (pg/mg protein) 9.6 ± 1.7 3.07 ± 0.2 ** 7.4 ± 1.1 ## Abv. BUN: blood urea nitrogen; IL-6: interleukin-6; KIM-1: kidney injury molecule-1; MDA: malondialdehyde; NGAL: neutrophil gelatinase-associated lipocalin; Nrf2: nuclear factor erythroid 2–related factor 2; SIRT-1: sirtuin-1; TNF-α: tumor necrosis factor-alpha. Results were presented as mean ± SEM. *p<0.01, ** p<0.001 (different from control group); # p<0.05, ## p<0.001 (different from Colistin and tap water group). Discussion In this experimental study, we investigated the nephroprotective potential of wogonin in a rat model of CIN. Colistin, administered intraperitoneally at 20 mg/kg, successfully induced AKI, as reflected by elevated plasma levels of BUN, creatinine, KIM-1, NGAL, and MDA, along with pronounced tubular necrosis and inflammation. Wogonin, given at 50 mg/kg/day for 10 days with oral gavage, significantly mitigated these effects, reducing functional (BUN, creatinine) and injury markers (KIM-1, NGAL), attenuating oxidative stress and inflammatory mediators (MDA, TNF-α, IL-6), and restoring antioxidant tissue markers (SIRT-1, Nrf2). These effects were accompanied by notable histological improvement, including reductions in tubular epithelial necrosis, luminal debris, and tubular dilatation. To the best of our knowledge, this is the first report demonstrating the nephroprotective role of wogonin in a CIN model. The primary finding of this study was the significant histopathological improvement observed in the colistin + wogonin group, underscoring its potent nephroprotective effects against CIN. Notable reductions in tubular epithelial necrosis, luminal debris accumulation, and tubular dilatation were evident, indicating preservation of renal microarchitecture and suggesting attenuation of tissue-level injury. These histological improvements are consistent with previous studies in diabetic nephropathy, cisplatin-induced nephropathy and ureteral obstruction models, where wogonin attenuated renal injury through modulation of the PI3K/Akt/NF-κB signaling axis and suppression of fibrotic and apoptotic processes 9,10,12,15,21,22 . Furthermore, wogonin’s ability to inhibit the TGF-β/Smad3 pathway, an established mediator of renal fibrosis, has been demonstrated in diabetic nephropathy, reinforcing its antifibrotic potential 9 . Consistent with literature, the structural improvements observed in our histological findings, likely reflecting the antifibrotic properties of wogonin, corroborate the biochemical results and further emphasize the therapeutic potential of wogonin in maintaining renal integrity under nephrotoxic stress. Nevertheless, fibrotic remodeling was not specifically assessed using dedicated markers such as Masson's trichrome staining or alpha-smooth muscle actin (α-SMA) immunohistochemistry. Considering the known anti-fibrotic actions of wogonin, future studies should incorporate fibrosis-specific endpoints to comprehensively evaluate its long-term structural benefits. Elevated plasma levels of BUN and creatinine in the colistin + tap water group confirmed significant renal impairment and validated the successful establishment of the nephrotoxicity model. However, these conventional markers may not fully capture early or localized tubular injury. To overcome this limitation, we also measured plasma levels of KIM-1 and NGAL, biomarkers that are more sensitive to tubular epithelial damage. KIM-1 reflects proximal tubular injury, while NGAL is associated with both acute tubular damage and inflammatory responses 23,24 . The significant reductions in these biomarkers following wogonin treatment suggest substantial attenuation of renal injury at both functional and cellular levels. Meng et al. 12 reported that wogonin alleviated cisplatin-induced AKI by suppressing pro-inflammatory cytokines and oxidative stress, leading to decreased serum BUN, creatinine and KIM-1 levels. Similarly, Badawy et al. 14 demonstrated that wogonin reduced serum BUN, creatinine by inhibiting NF-κB and MAPK pathways in cisplatin-induced AKI models. In nephrotoxicity models in the literature, the nephroprotective effects of wogonin have generally been evaluated using only traditional functional biomarkers. Thus, the inclusion and modulation of early-response injury biomarkers such as NGAL and KIM-1, as demonstrated in our study, provide an important contribution to the literature by enabling more sensitive detection and evaluation of renal injury. With previous similar nephrotoxicity patterns, CIN represents a well-characterized and clinically significant form of drug-induced renal injury, primarily mediated through oxidative stress, inflammation, and apoptosis 2,3,5,8,25 . In the present study, significant elevations in plasma TNF-α, IL-6, and MDA levels following colistin exposure reflect the activation of systemic inflammatory and oxidative stress pathways. Wogonin treatment effectively attenuated these aberrations, indicating its potential role in modulating redox homeostasis and inflammatory signaling. These effects are supported by earlier experimental studies indicating that wogonin exerts anti-inflammatory and antioxidant activity by suppressing nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling cascades 9,10,13,15 . Although no prior studies have investigated wogonin in a CIN model, Badawy et al. 14 demonstrated in a cisplatin-induced nephrotoxicity model that wogonin pretreatment significantly attenuated renal expression of IL-1β, TNF-α, and NF-κB, emphasizing its potent anti-inflammatory properties in renal injury settings. Similarly, Zheng et al. 9 reported that wogonin attenuated renal inflammation and fibrosis in a diabetic nephropathy model by reducing proinflammatory cytokines, including TNF-α and IL-1β, through inhibition of the NF-κB and TGF-β1/Smad3 signaling pathways. Mechanistically, wogonin has been shown to inhibit the nuclear translocation of NF-κB and suppress the expression of inducible nitric oxide synthase (iNOS), both of which are important mediators in the propagation of proinflammatory signaling pathways 26,27 . These molecular actions may underlie the nephroprotective effects observed in our study. However, while our findings support the functional and histopathological efficacy of wogonin, they are limited by the absence of direct molecular analyses of inflammatory and apoptotic pathways. This gap restricts the mechanistic resolution of the observed effects. Future investigations should incorporate pathway-specific analyses, including necroptosis, autophagy, and apoptosis-related signaling, to elucidate the precise molecular pathways of wogonin’s nephroprotective activity in CIN models. SIRT-1 and Nrf2 are substantial regulators of cellular stress responses, apoptosis, and antioxidant defense mechanisms. In the context of AKI, both molecules serve as critical modulators of renal cell survival and protection against oxidative damage 28–30 . In our study, wogonin administration resulted in a significant upregulation of SIRT-1 and Nrf2 expression in renal tissue, indicating activation of endogenous cytoprotective pathways. The restoration of SIRT-1 expression is particularly relevant, given its known role in promoting mitochondrial function, regulating apoptotic signaling, and attenuating inflammation 28,29 . Previous studies have demonstrated that wogonin upregulates Nrf2 expression in various models of tissue injury, including acute lung injury, cardiac injury, hepatic damage, ischemia-reperfusion injury, traumatic brain injury, and cancer 27,31–39 . This upregulation has been shown to inhibit pro-apoptotic Bcl-2-associated X protein (Bax) signaling and enhance B-cell lymphoma 2 (Bcl-2) mediated autophagy, thereby promoting cellular survival under oxidative stress 40 . Also, Nrf2 activation by wogonin supports its antioxidative capacity through the induction of downstream effectors such as heme oxygenase-1 (HO-1), thereby enhancing the renal antioxidant defense system and neutralizing reactive oxygen species 27,30,32 . In this context, our findings provide a valuable contribution to the existing literature, as one of the few studies to suggest that the protective renal effects of wogonin in a nephrotoxicity model. The concurrent upregulation of SIRT-1 and Nrf2 observed in our study implies a synergistic mechanism by which wogonin alleviates oxidative stress, inhibits apoptotic signaling, and supports the preservation of tubular epithelial integrity. However, despite demonstrating increased expression of these regulators, our study did not include direct evaluation of downstream effectors such as HO-1, the Bax/Bcl-2 ratio, or cleaved caspase-3, critical mediators in oxidative stress and apoptosis signaling. To clarify the mechanistic underpinnings of wogonin's nephroprotective effects in the context of CIN, future investigations should incorporate molecular approaches such as Western blotting or immunohistochemical analysis to characterize the downstream signaling events associated with SIRT-1 and Nrf2 activation. This study presents several notable strengths. It is among the first to comprehensively evaluate the nephroprotective potential of wogonin in a well-established model of CIN, integrating systemic and renal-specific biomarkers with rigorous histopathological assessment. The experimental design included controlled conditions, blinding during histological scoring, and the inclusion of both functional and injury-related markers, enhancing the validity and reproducibility of the findings. Importantly, the observed histological protection, characterized by reduced tubular necrosis and architectural preservation, provides structural corroboration of the biochemical improvements, reinforcing the translational relevance of wogonin in clinical scenarios where colistin use is necessary but nephrotoxicity is a limiting factor. Nevertheless, several limitations must be acknowledged. The study did not include direct measurements of glomerular filtration rate (GFR), which could have offered a more precise functional assessment. This omission is primarily due to the technical challenges associated with urine collection in rodents. Additionally, the investigation was limited to a single dosing regimen (50 mg/kg/day), selected based on prior literature. However, the absence of a dose–response analysis limits our ability to determine the optimal therapeutic window or identify potential toxicity thresholds. Moreover, although significant phenotypic improvements were documented, the study did not incorporate molecular analyses to elucidate the precise signaling pathways involved in wogonin’s mechanism of action. Future studies should aim to overcome these limitations by incorporating molecular tools to investigate necroptosis, autophagy, apoptosis, and fibrosis-related signaling. Dose–response studies and exploration of synergistic effects with existing nephroprotective agents across diverse nephrotoxicity models will further clarify the translational potential of wogonin as an adjunctive therapeutic strategy. Conclusion Wogonin demonstrated significant nephroprotective efficacy in a rat model of CIN through the attenuation of oxidative stress, suppression of pro-inflammatory cytokine expression, and amelioration of tubular epithelial damage. These effects were characterized by histopathological improvements, and reductions in inflammatory and oxidative biomarkers, such as MDA, TNF-α, and IL-6, accompanied by the upregulation of key cytoprotective mediators, including SIRT-1 and Nrf2. These findings underscore the need for clinical evaluation of wogonin as a promising adjunctive therapy to protect renal function in contexts necessitating colistin use. Abbreviations AKI: Acute kidney injury Akt: Protein Kinase B α-SMA: Alpha-smooth muscle actin ANOVA: One-way analysis of variance Bax: Bcl-2-associated X protein Bcl2: B-cell lymphoma 2 BUN: Blood urea nitrogen CIN: Colistin-induced nephrotoxicity ELISA: Enzyme-linked immunosorbent assay GFR: Glomerular filtration rate H&E: Hematoxylin and eosin HO-1: Heme oxygenase-1 IL-1β: Interleukin-1 beta IL-6: Interleukin-6 iNOS: Inducible nitric oxide synthase KIM-1: Kidney injury molecule-1 MAPK: Mitogen-activated protein kinase MDA: Malondialdehyde NF-κB: Nuclear factor-kappa B NGAL: Neutrophil gelatinase-associated lipocalin nM: Nanomoles Nrf2: Nuclear factor erythroid 2–related factor 2 PBS: Phosphate-buffered saline PI3K: Phosphatidylinositol 3-Kinase SEM: Standard error of the mean SIRT-1: Sirtuin-1 Smad3: Mothers against decapentaplegic homolog 3 TBARS: Thiobarbituric acid reactive substances TGF-β: Transforming Growth Factor-beta TNF-α: Tumor necrosis factor-alpha Declarations Acknowledgements None Authors Contribution A.S.T., M.A.E. and O.E. performed the animal experiments, including group allocation, treatment administration, and sample collection. A.S.T., B.E.G. and M.A.C assisted in experimental design, manuscript revision, and contributed to the interpretation of results. M.A.E. and O.E. conducted the biochemical analyses and helped prepare figures and tables. The authors confirm that no paper mill and artificial intelligence was used. All authors contributed to the study design, read, and approved the final manuscript, and agree to be accountable for all aspects of the work. Funding None Availability of Data and Materials Due to institutional privacy policies and ethical considerations, the datasets generated and analyzed during the current study are not publicly available but can be made available upon reasonable request to the corresponding author. Ethics This study was approved by the Institutional Animal Care and Ethical Committee of the Science University, Istanbul (Ethics Approval Number: 2925042821). All procedures involving animals were conducted in full compliance with the internationally recognized ARRIVE 2.0 guidelines and were designed to adhere strictly to the principles of replacement, reduction, and refinement (the 3Rs). Conflict and Interest The authors declare no conflicts of interest related to this study. References Tsuji, B. T. et al. International Consensus Guidelines for the Optimal Use of the Polymyxins: Endorsed by ACCP, ESCMID, IDSA, ISAP, SCCM, and SIDP. Pharmacotherapy. 39, 10–39 (2019). Chien, H. T., Lin, Y. C., Sheu, C. C., Hsieh, K. P. & Chang, J. S. Is colistin-associated acute kidney injury clinically important in adults? A systematic review and meta-analysis. Int. J. Antimicrob. Agents. 55, 105889 (2020). Gai, Z., Samodelov, S. L., Kullak-Ublick, G. A. & Visentin, M. Molecular mechanisms of colistin-induced nephrotoxicity. Molecules. 24, 653 (2019). Falagas, M. E. et al. Clinical use of intravenous polymyxin B for multidrug-resistant Gram-negative infections: An evaluation of the current evidence. J. Glob. Antimicrob. Resist. 24, 342–359 (2021). Ordooei Javan, A., Shokouhi, S. & Sahraei, Z. A review on colistin nephrotoxicity. Eur. J. Clin. Pharmacol. 71, 801–810 (2015). Bintang, M. A. K. M., Nopparat, J. & Srichana, T. In vivo evaluation of nephrotoxicity and neurotoxicity of colistin formulated with sodium deoxycholate sulfate in mice. Naunyn Schmiedebergs Arch. Pharmacol. 396, 3243–3252 (2023). Jafari, F. & Elyasi, S. Prevention of colistin-induced nephrotoxicity: a review of preclinical and clinical data. Expert Rev. Clin. Pharmacol. 14, 1113–1131 (2021). Heybeli, C., Oktan, M. A. & Çavdar, Z. Rat models of colistin nephrotoxicity: previous experimental research and future perspectives. Eur. J. Clin. Microbiol. Infect. Dis. 38, 1387–1393 (2019). Zheng, Z. C. et al. Wogonin ameliorates renal inflammation and fibrosis by inhibiting NF-κB and TGF-β1/Smad3 pathways in diabetic nephropathy. Drug Des. Devel. Ther. 14, 4135–4148 (2020). Lei, L. et al. Wogonin alleviates kidney tubular epithelial injury in diabetic nephropathy by inhibiting PI3K/Akt/NF-κB signaling. Drug Des. Devel. Ther. 15, 3131–3150 (2021). Talbi, A. et al. Pharmacokinetics, tissue distribution, excretion and plasma protein binding studies of wogonin in rats. Molecules. 19, 5538–5549 (2014). Meng, X. M. et al. Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis. Lab Invest. 98, 79–94 (2018). Liu, Y. et al. Wogonin upregulates SOCS3 to alleviate injury in diabetic nephropathy by inhibiting TLR4-mediated JAK/STAT/AIM2 signaling. Mol. Med. 30, 78 (2024). Badawy, A. M. et al. Wogonin pre-treatment attenuates cisplatin-induced nephrotoxicity in rats: Impact on PPAR-γ, inflammation, apoptosis, and Wnt/β-catenin. Chem. Biol. Interact. 308, 137–146 (2019). Kuo, H. L. et al. Wogonin ameliorates ER stress-associated inflammation, apoptosis and fibrosis in unilateral ureteral obstruction in mice. Eur. J. Pharmacol. 977, 176676 (2024). Percie du Sert, N. et al. Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. 18, e3000411 (2020). Tannenbaum, J. & Bennett, B. T. Russell and Burch’s 3Rs then and now: The need for clarity in definition and purpose. J. Am. Assoc. Lab. Anim. Sci. 54, 120 (2005). Sivanesan, S. S. et al. Gelofusine ameliorates colistin-induced nephrotoxicity. Antimicrob. Agents Chemother. 61, e00985-17 (2017). Erbas, O. et al. Oxytocin alleviates cisplatin-induced renal damage in rats. Iran. J. Basic Med. Sci. 17, 747–752 (2014). Faul, F., Erdfelder, E., Lang, A. G. & Buchner, A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods. 39, 175–191 (2007). Wang, Y. et al. Wogonin induces apoptosis and reverses sunitinib resistance in renal carcinoma via CDK4-RB inhibition. Front. Pharmacol. 11, 1152 (2020). Li, J. et al. Impact of baicalin and Scutellaria baicalensis components on renal fibrosis in diabetic kidney disease. Front. Pharmacol. 15, 1480626 (2024). Vaidya, V. S. et al. Kidney injury molecule-1 outperforms traditional kidney biomarkers in preclinical studies. Nat. Biotechnol. 28, 478–485 (2010). Haase, M. et al. Accuracy of NGAL in diagnosis and prognosis of acute kidney injury: A meta-analysis. Am. J. Kidney Dis. 54, 1012–1024 (2009). Dai, C. et al. Colistin-induced nephrotoxicity involves mitochondrial, death receptor, and ER pathways in mice. Antimicrob. Agents Chemother. 58, 4075–4085 (2014). Yao, J. et al. Wogonin prevents LPS-induced acute lung injury via PPARγ-mediated NF-κB inhibition. Immunology. 143, 241–257 (2014). He, X. et al. Wogonin attenuates inflammation and oxidative stress in mastitis by Akt/NF-κB inhibition and Nrf2/HO-1 activation. Cell Stress Chaperones. 28, 989–999 (2023). Albalawi, R. S. et al. Parthenolide phytosomes attenuate gentamicin-induced nephrotoxicity via SIRT-1, Nrf2, HO-1, and NQO1. Molecules. 28, 2741 (2023). Kumari, A., Sodum, N., Ravichandiran, V. & Kumar, N. Role of SIRT-1 in treatment and prevention of diabetic nephropathy: A review. Curr. Mol. Pharmacol. 16, 811–831 (2023). Zaghloul, R. A., Abdelghany, A. M. & Samra, Y. A. Rutin and selenium nanoparticles protect against diabetic nephropathy by modulating Jak-2/Stat3 and Nrf2/HO-1. Eur. J. Pharmacol. 933, 175289 (2022). Zhou, Y., Dou, F., Song, H. & Liu, T. Wogonin exerts anti-ulcerative effects in colitis via Nrf2/TLR4/NF-κB pathway. Environ. Toxicol. 37, 954–963 (2022). Feng, Y. et al. Wogonin protects against brain injury by reducing oxidative stress via PI3K/Nrf2/HO‑1 pathway. Int. J. Mol. Med. 49, 53 (2022). Liu, X. et al. Wogonin induces ferroptosis in pancreatic cancer via Nrf2/GPX4 axis inhibition. Front. Pharmacol. 14, 1129662 (2023). Dai, J. M. et al. Wogonin alleviates liver injury in sepsis through Nrf2-mediated NF-κB suppression. J. Cell. Mol. Med. 25, 5782–5798 (2021). Yang, H. et al. Wogonin attenuates inflammation and oxidative stress in acute lung injury via PPARα, AKT, and NRF2 pathways. Naunyn Schmiedebergs Arch. Pharmacol. (2025). Cheng, Z. et al. Wogonin alleviates NLRP3 inflammasome activation after cerebral ischemia-reperfusion via AMPK/SIRT1. Brain Res. Bull. 207, 110886 (2024). Ge, J., Yang, H., Yu, N., Lin, S. & Zeng, Y. Wogonin alleviates sepsis-induced lung injury by modulating macrophage polarization via SIRT1–FOXO1. Tissue Cell. 88, 102400 (2024). Wang, L. et al. Cardiac protection by wogonin in pulmonary fibrosis via Sirt1/γ-H2AX pathway. Front. Pharmacol. 16, 1551141 (2025). Qian, C. et al. Wogonin enhances ROS-induced apoptosis and potentiates chemo effects via Nrf2 suppression in HepG2 cells. Free Radic. Res. 48, 607–621 (2014). Liu, X. Q. et al. Wogonin protects podocytes via Bcl-2-mediated autophagy and apoptosis inhibition in diabetic kidney disease. Acta Pharmacol. Sin. 43, 96–110 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviews received at journal 30 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers invited by journal 14 Aug, 2025 Editor assigned by journal 08 Aug, 2025 Submission checks completed at journal 18 Jul, 2025 First submitted to journal 18 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7124382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":502510183,"identity":"a43ac16a-ea64-4b05-b4e3-8db4b9432f10","order_by":0,"name":"Ahmet Salih Tüzen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYBADOTYG5gYGBjaiFDODSWM2BkYStSQ2EK2Fv//8MckfFXfS+9gPNj7mKWOQ5xc7gF+LxI1kNgmJM89y23gSm415zjEYzpydQMCaG8xsEoZth3PbJBjbpHnbGBIMbhPQIn/+MJtE4r/D6WxEazE4AHTYwYbDCcRrMbyRbGzZcOywIcgvhnPOSRD2i9z5gw9v/qg5LC/ffvjggzdlNvL80gS0oAMJ0pSPglEwCkbBKMAOALE1PRmnGekjAAAAAElFTkSuQmCC","orcid":"","institution":"Ministry of Health İzmir Katip Çelebi University Atatürk Education and Research Hospital","correspondingAuthor":true,"prefix":"","firstName":"Ahmet","middleName":"Salih","lastName":"Tüzen","suffix":""},{"id":502510184,"identity":"990930db-f613-4065-bc95-b9c9d84b4b2e","order_by":1,"name":"Birzat Emre Gölboyu","email":"","orcid":"","institution":"Ministry of Health İzmir Katip Çelebi University Atatürk Education and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Birzat","middleName":"Emre","lastName":"Gölboyu","suffix":""},{"id":502510185,"identity":"0407fa63-1e47-42b6-be0a-f93f3743b1e6","order_by":2,"name":"Mehmet Ali Coşar","email":"","orcid":"","institution":"Ministry of Health İzmir Katip Çelebi University Atatürk Education and Research Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"Ali","lastName":"Coşar","suffix":""},{"id":502510187,"identity":"0713637e-babe-4185-b976-4b0f1c088b85","order_by":3,"name":"Mümin Alper Erdoğan","email":"","orcid":"","institution":"Izmir Katip Celebi University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mümin","middleName":"Alper","lastName":"Erdoğan","suffix":""},{"id":502510189,"identity":"1140aea2-0a90-4dbb-8937-193f2228b798","order_by":4,"name":"Oytun Erbaş","email":"","orcid":"","institution":"Biruni University Faculty of Medicine, BAMER","correspondingAuthor":false,"prefix":"","firstName":"Oytun","middleName":"","lastName":"Erbaş","suffix":""}],"badges":[],"createdAt":"2025-07-14 20:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7124382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7124382/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89599743,"identity":"f86f676c-93f7-494a-bb8b-c7313cab597a","added_by":"auto","created_at":"2025-08-21 17:48:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79013,"visible":true,"origin":"","legend":"\u003cp\u003eThe Experimental Study Flow Chart\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7124382/v1/38580454a520bc561c7a0990.jpg"},{"id":89599744,"identity":"d592c2b9-946c-4d98-963e-d5bcf5aa9860","added_by":"auto","created_at":"2025-08-21 17:48:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":301096,"visible":true,"origin":"","legend":"\u003cp\u003eThe Kidney Histopathological Sections From Groups (H\u0026amp;E Staining, ×20 and ×40 Magnification)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7124382/v1/f300e1d8804495d24397c4ce.jpg"},{"id":89600469,"identity":"af1d51fa-6049-465d-a2c2-5aa4dcc4cf9e","added_by":"auto","created_at":"2025-08-21 18:04:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":862961,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7124382/v1/df059c91-f7aa-46aa-aa8a-48561cda48c7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective Effect of Wogonin Against Colistin-Induced Nephrotoxicity in Wistar Rats: A Controlled Experimental Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolymyxins, particularly colistin, have re-emerged as last-resort antibiotics for treating multidrug-resistant Gram-negative bacterial infections, especially in critically ill patients. Despite its clinical significance, the therapeutic utility of colistin is greatly restricted due to its nephrotoxic potential, which affects approximately 20–50% of treated patients in a dose- and duration-dependent manner \u003csup\u003e1–4\u003c/sup\u003e. The nephrotoxicity primarily manifests as acute kidney injury (AKI), involving oxidative stress, inflammation, and tubular epithelial damage \u003csup\u003e3,5\u003c/sup\u003e. Experimental models have consistently shown that colistin induces oxidative stress, mitochondrial dysfunction, lipid peroxidation, and apoptosis, leading to characteristic histological alterations such as tubular epithelial necrosis, interstitial inflammation, and loss of brush border integrity. These pathophysiological mechanisms, confirmed by elevations in plasma creatinine, blood urea nitrogen (BUN), and lipid peroxidation markers such as malondialdehyde (MDA) levels, underline its potent nephrotoxic profile \u003csup\u003e6–8\u003c/sup\u003e. Even in clinical scenarios where colistin remains indispensable, its use is often approached with caution due to the high likelihood of renal complications; particularly in vulnerable, critically ill populations with limited renal reserve \u003csup\u003e1\u003c/sup\u003e. In this context, there remains a critical and urgent need for effective adjunctive therapies that can protect the kidneys from CIN without compromising its potent antimicrobial activity \u003csup\u003e1,2,4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmong various mechanistic contributors to CIN, pro-inflammatory mediators such as interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) play important roles in propagating renal inflammation and injury \u003csup\u003e9,10\u003c/sup\u003e. Within this pathophysiological framework, natural compounds possessing antioxidant and anti-inflammatory properties have garnered increasing interest as promising adjunctive therapies for mitigating nephrotoxic insults. Wogonin (5,7-dihydroxy-8-methoxy flavone), a natural flavonoid derived from the roots of \u003cem\u003eScutellaria baicalensis\u003c/em\u003e, has attracted growing interest due to its diverse pharmacological properties, particularly its antioxidant, anti-inflammatory, and anti-fibrotic effects \u003csup\u003e11\u003c/sup\u003e. These attributes have positioned it as a promising candidate in experimental models of nephrotoxicity. In various preclinical studies, wogonin has been shown to exert nephroprotective effects by modulating critical molecular pathways, including the inhibition of NF-κB, Transforming Growth Factor-beta (TGF-β) / Mothers against decapentaplegic homolog 3 (Smad3), and Phosphatidylinositol 3-Kinase (PI3K) / Protein Kinase B (Akt) signaling, while enhancing antioxidant defenses through activation of the Nuclear factor erythroid 2–related factor 2 (Nrf2) / Heme oxygenase-1 (HO-1) pathway. Additionally, it reduces the expression of key pro-inflammatory cytokines such as TNF-α and Interleukin-6 (IL-6), thereby attenuating renal inflammation, oxidative stress, and fibrotic remodeling \u003csup\u003e9–13\u003c/sup\u003e. These mechanisms have been validated in several kidney injury models, including diabetic nephropathy, cisplatin-induced AKI, and unilateral ureteral obstruction, where wogonin consistently demonstrated protective effects on renal structure and function \u003csup\u003e9,10,12–15\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite its broad-spectrum nephroprotective actions in various experimental models, no published studies have yet systematically evaluated the potential protective effects of wogonin in CIN. Given that CIN is mediated by distinct injury mechanisms, such as mitochondrial dysfunction, lipid peroxidation, and pro-inflammatory signaling, translating wogonin’s known nephroprotective mechanisms to this specific context is of high clinical relevance \u003csup\u003e5,8\u003c/sup\u003e. The lack of histopathological and biochemical studies investigating the effects of wogonin in CIN models demonstrate a significant gap in the current literature. To address this, we designed a randomized controlled experimental study using adult female Wistar rats to evaluate the nephroprotective efficacy of wogonin against CIN. Our primary hypothesis was that wogonin, through its modulation of oxidative stress and inflammatory pathways, would mitigate renal injury and preserve renal function and morphology. To investigate this, the present study primarily focused on the histopathological assessment of renal tissue using detailed semiquantitative scoring. As secondary objectives, we evaluated a comprehensive panel of renal biomarkers in both serum and tissue samples, including functional markers: BUN, creatinine; early injury biomarkers: Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL); inflammatory mediators: MDA, IL-6, and TNF-α, and oxidative stress-related regulators: Sirtuin-1 (SIRT-1) and Nrf2. These parameters were selected to explore the mechanistic basis of wogonin’s potential nephroprotective activity. Accordingly, this study aims to investigate the nephroprotective efficacy of wogonin as a adjunctive therapeutic agent against CIN using a randomized in vivo rat model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eEthical Considerations\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals were reviewed and approved by the Institutional Animal Care and Ethical Committee of the Science University, Istanbul (Approval Number: 2925042821), and conducted in full compliance with the internationally recognized ARRIVE 2.0 guidelines \u003csup\u003e16\u003c/sup\u003e. All methods were performed in accordance with the relevant guidelines and regulations. The study was meticulously designed to ensure the ethical treatment of animals, with particular emphasis on the principles of replacement, reduction, and refinement (the 3Rs) \u003csup\u003e17\u003c/sup\u003e. Every effort was made to minimize pain, distress, and suffering throughout the experimental protocol. All animals were closely monitored during the study period to ensure humane care and early intervention in the case of any adverse effects.\u003c/p\u003e\n\u003cp\u003eStudy Design and Animals\u003c/p\u003e\n\u003cp\u003eThis controlled in vivo experimental study was conducted using 30 adult female Wistar rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e), each weighing between 200–210 grams and aged 8-10 weeks, obtained from the Central Animal Facility of Science University, Istanbul, Turkey. Upon procurement, all animals underwent a one-week acclimatization period to minimize environmental stress and allow for physiological and behavioral stabilization. Animals were housed in standard ventilated polycarbonate cages, five rats per cage, under controlled environmental conditions; ambient temperature of 22 ± 2 °C, relative humidity maintained at 50-60%, and a 12-hour light/dark cycle. Environmental enrichment strategies, including nesting materials and shelter objects, were uniformly applied to promote natural behaviors and improve welfare. Rats were provided \u003cem\u003ead libitum\u003c/em\u003e access to standardized pellet chow and filtered tap water for the entire duration of the study. Each animal was examined daily for clinical signs of illness or distress, including monitoring for changes in body weight, grooming behavior, food and water intake, and overall activity. Humane endpoints were established prior to study initiation and were strictly enforced to prevent unnecessary suffering.\u003c/p\u003e\n\u003cp\u003eExperimental Groups and Treatment Protocol\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven the complex pathophysiology of CIN, encompassing oxidative stress, inflammation, apoptosis, and structural renal damage, in vivo models are indispensable. Accordingly, our study employed an animal model to assess pharmacokinetic and pharmacodynamic responses, immunological effects, and renal structural changes following colistin exposure and wogonin treatment. The treatment regimen and experimental protocol were designed to evaluate the therapeutic efficacy of wogonin in attenuating CIN. A total of 30 adult female Wistar rats were enrolled in the study and randomly assigned into three groups (n=10 per group) to minimize selection bias. First group served as the normal control group and received no pharmacological intervention during the study period. The second group (colistin + tap water group) and the third group (colistin + wogonin group) were administered a single intraperitoneal injection of colistin at a dose of 20 mg/kg to induce AKI. Following colistin (Kocak Pharma, Istanbul, Turkey) administration, colistin + tap water group rats received 1 mL/kg/day of tap water via oral gavage for 10 consecutive days, while colistin + wogonin group received wogonin (Adipogen, San Diego, CA, USA) at a dose of 50 mg/kg/day via oral gavage for the same duration. Colistin dosage was selected based on prior studies demonstrating reproducible AKI in rodent models \u003csup\u003e6,18\u003c/sup\u003e. The dosage and administration method for wogonin were selected based on previous preclinical studies demonstrating its nephroprotective effects in rodent models of renal injury \u003csup\u003e15\u003c/sup\u003e. During the treatment period, two animals from colistin + tap water group died, presumably due to severe CIN. These animals were excluded from subsequent biochemical and histological analyses. No mortality was observed in either the control group or the wogonin-treated group.\u003c/p\u003e\n\u003cp\u003eStudy Outcomes\u003c/p\u003e\n\u003cp\u003eThe primary outcome of this study was the evaluation of renal histopathological alterations using semiquantitative scoring to determine the extent of tubular epithelial damage, including necrosis, tubular dilatation, and luminal debris, in response to colistin and the potential protective effect of wogonin. The secondary outcomes included assessment of renal function through biochemical markers such as BUN and serum creatinine, early tubular injury biomarkers including KIM-1 and NGAL, and systemic and tissue-specific inflammatory and oxidative stress parameters. These included MDA, TNF-α, IL-6, and antioxidant regulators such as SIRT-1 and Nrf2. These outcome measures were selected to comprehensively evaluate the pathophysiological impact of colistin and to determine the nephroprotective efficacy of wogonin at both functional and molecular levels.\u003c/p\u003e\n\u003cp\u003eSample Collection and Sacrifice\u003c/p\u003e\n\u003cp\u003eAt the conclusion of the 10-day treatment period, all surviving animals were anesthetized using a high-dose intraperitoneal injection of ketamine (100 mg/kg) (Vemilac, Istanbul, Turkey) and xylazine (10 mg/kg) (Bioveta, Ivanovice na Hané, Czech Republic) to ensure deep surgical anesthesia prior to tissue and blood collection. Following confirmation of adequate anesthesia, the kidneys were rapidly excised, rinsed with ice-cold saline to remove residual blood, and processed for both biochemical and histopathological examinations. One portion of each kidney was fixed in 10% buffered formalin for histological analysis, while the remaining tissue was snap-frozen and stored at −20 °C for subsequent biochemical assays. Blood samples were obtained via cardiac puncture using a sterile 1 mL syringe and immediately transferred into heparinized collection tubes for biochemical analysis. Subsequently, the animals were sacrificed by blood collection with cardiac puncture, in accordance with the predefined humane endpoints and ethical guidelines.\u003c/p\u003e\n\u003cp\u003eBiochemical, Oxidative Stress Parameters, and Histopathological Evaluation\u003c/p\u003e\n\u003cp\u003ePlasma samples were obtained by centrifugation of collected blood at 3000 rpm for 10 minutes at room temperature. The separated plasma was aliquoted and stored at −20 °C until analysis. Renal function was evaluated by measuring plasma levels of BUN and creatinine using a Beckman-Coulter AU 640 automated analyzer (Beckman-Coulter Inc., CA, USA). To assess systemic inflammation, plasma concentrations of IL-6, TNF-α, as well as the tubular damage biomarkers KIM-1 and NGAL, were quantified using rat-specific enzyme-linked immunosorbent assay (ELISA) kits (Sigma-Aldrich, St. Louis, MO, USA).\u003c/p\u003e\n\u003cp\u003eOxidative stress was assessed by measuring MDA concentrations, an indicator of lipid peroxidation, using the thiobarbituric acid reactive substances (TBARS) method with MDA assay kit (Sigma-Aldrich, St. Louis, MO, USA). MDA concentrations were expressed in nanomoles (nM), with tetraethoxypropane used for calibration. Trichloroacetic acid and TBARS reagent were added to plasma samples and incubated at 100 °C for 60 minutes. After cooling on ice, the samples were centrifuged at 3000 rpm for 20 minutes, and the absorbance of the supernatant was read at 535 nm using a spectrophotometer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor renal tissue analysis, whole kidneys were homogenized in phosphate-buffered saline (pH=7.4) at a ratio of 1:5 (w/v) using a glass homogenizer. Homogenates were centrifuged at 5000 × g for 15 minutes, and supernatants were collected for biochemical evaluation. Total protein concentration was determined using the Bradford assay, with bovine serum albumin as the reference standard. Tissue levels of SIRT-1 and Nrf2 were measured from homogenates using rat-specific ELISA kits (Sigma-Aldrich, St. Louis, MO, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor histopathological evaluation, kidney tissues were fixed in 10% neutral-buffered formalin for 48 hours, embedded in paraffin blocks, and sectioned at 4 μm thickness. Sections were stained with hematoxylin and eosin (H\u0026amp;E) and examined under light microscopy. All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope (Olympus Co., Tokyo, Japan). Morphological assessments were performed using a computerized image analysis system (Image-Pro Express 1.4.5, Media Cybernetics, Rockville, MD, USA) across 10 non-overlapping fields per section at ×20 magnification. The examiner was blinded to the treatment groups to ensure objectivity. A semi-quantitative scoring system was used to evaluate the degree of tubular epithelial necrosis, luminal necrotic debris, tubular dilatation, and interstitial inflammation. The scoring scale was as follows: 0 (0–5%), 1 (6–20%), 2 (21–40%), 3 (41–60%), 4 (61–80%), and 5 (81–100%) \u003csup\u003e19\u003c/sup\u003e. Composite injury scores were computed to reflect the overall histological damage for each rat.\u003c/p\u003e\n\u003cp\u003eStatistical Analysis\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). The normality of data distribution was assessed using the Shapiro-Wilk test to determine the appropriate statistical method for each variable. For parametric data, comparisons between groups were performed using Student’s t-test or one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple intergroup comparisons when indicated. For non-parametric data, the Mann–Whitney U test was used to compare differences between groups. All data are expressed as mean ± standard error of the mean (SEM). A \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.05 was considered statistically significant for all analyses.\u003c/p\u003e\n\u003cp\u003eA priori power analysis was performed using G*Power version 3.1.9.6 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) \u003csup\u003e20\u003c/sup\u003e to determine the minimum number of animals required per group to detect statistically significant differences among experimental groups. The analysis was informed by histopathological evaluation from previous preclinical studies investigating nephroprotective interventions in AKI models, using a Cohen’s effect size of 0.8, a significance level (α) of 0.05, and a statistical power (1−β) of 0.95. Based on these parameters, the estimated sample size for one-way ANOVA with post hoc comparisons was calculated to be at least eight rats per group. To accommodate potential attrition due to CIN, we included 10 animals in each group, accounting for an anticipated dropout rate of approximately 20%.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe experimental study flow chart and group distribution are outlined in Fig 1. A total of 30 adult female Wistar rats were initially enrolled. Two rats from the colistin + tap water group died during the 10-day treatment period and were excluded from all analyses. No mortality was observed in either the normal control group or the colistin + wogonin group.\u003c/p\u003e\n\u003cp\u003eHistopathological evaluation of renal injury are summarized in Table 1. Histopathological scores for tubular epithelial necrosis, luminal necrotic debris, and tubular dilatation were higher in the colistin + tap water group compared to the control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). These scores were lower in the colistin + wogonin group than in the colistin + tap water group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). However, no significant differences in interstitial inflammation scores were observed across the groups (Table 1). The kidney histopathological sections are shown in Fig 2, including H\u0026amp;E Staining images from all three groups.\u003c/p\u003e\n\u003cp\u003eTable 1. Histopathological Evaluation of Renal Injury\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"621\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003eNormal control Group\u003c/p\u003e\n \u003cp\u003e(n=10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eColistin + Tap Water Group\u003c/p\u003e\n \u003cp\u003e(n=8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eColistin + Wogonin Group\u003c/p\u003e\n \u003cp\u003e(n=10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 621px;\"\u003e\n \u003cp\u003eHistopathological score\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eTubular epithelial necrosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.1\u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e2.5 \u0026plusmn; 0.2 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.9 \u0026plusmn; 0.1 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eLuminal necrotic debris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.1 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.1 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e1.1 \u0026plusmn; 0.2 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eTubular dilatation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.2 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e1.7 \u0026plusmn; 0.3 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.8 \u0026plusmn; 0.3 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 186px;\"\u003e\n \u003cp\u003eInterstitial inflammation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e0.2 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.3 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.2 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults were presented as mean \u0026plusmn; SEM. *p\u0026lt;0.001, (different from control group); # p\u0026lt;0.01 (different from Colistin and tap water group).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasma levels of creatinine and BUN were measured to assess renal function. Both parameters were elevated in the colistin + tap water group relative to the normal control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). In the colistin + wogonin group, plasma creatinine and BUN levels were lower than those in the colistin + tap water group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Table 2). Also, early markers of renal injury, the plasma levels of KIM-1 and NGAL were higher in the colistin + tap water group compared to the normal control group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). These levels were reduced in the colistin + wogonin group relative to the colistin + tap water group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Table 2).\u003c/p\u003e\n\u003cp\u003eA key lipid peroxidation molecule, plasma MDA levels were elevated in the colistin + tap water group compared to controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Lower levels of MDA were observed in the colistin + wogonin group relative to the colistin + tap water group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Similarly, the pro-inflammatory cytokines, plasma concentrations of TNF-\u0026alpha; and IL-6 were also elevated in the colistin + tap water group (respectively, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Both markers were lower in the colistin + wogonin group than in the colistin + tap water group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRenal tissue levels of SIRT-1 and Nrf2 were lower in the colistin + tap water group than in the control group (respectively, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). These levels were higher in the colistin + wogonin group compared to the colistin + tap water group (respectively, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (Table 2).\u003c/p\u003e\n\u003cp\u003eTable 2. Plasma Biochemical Parameters and Renal Tissue Biomarkers\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"631\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eNormal control Group\u003c/p\u003e\n \u003cp\u003e(n=10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eColistin + Tap Water Group\u003c/p\u003e\n \u003cp\u003e(n=8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003eColistin + Wogonin Group\u003c/p\u003e\n \u003cp\u003e(n=10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 631px;\"\u003e\n \u003cp\u003eRenal Functional and Injury Biomarkers in Plasma\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eBUN (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e20.2 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e41.1 \u0026plusmn; 2.5 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e27.3 \u0026plusmn; 0.9 ##\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eCreatinine (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.38 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e1.17 \u0026plusmn; 0.2 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.74 \u0026plusmn; 0.1 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eKIM-1(pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e27.2 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e201.5 \u0026plusmn; 4.6 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e86.9 \u0026plusmn; 3.1 ##\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eNGAL (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e45.5 \u0026nbsp;\u0026plusmn; \u0026nbsp; \u0026nbsp; 3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e116.2 \u0026nbsp;\u0026plusmn; \u0026nbsp; \u0026nbsp; 5.9 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e67.6 \u0026nbsp;\u0026plusmn; \u0026nbsp; \u0026nbsp; 4.4 ##\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 631px;\"\u003e\n \u003cp\u003eOxidative Stress and Inflammatory Parameters\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003ePlasma MDA (nM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e38.7 \u0026plusmn; 2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e77.1 \u0026plusmn; 4.5 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e54.8 \u0026plusmn; 2.9 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003ePlasma TNF-alfa (pg/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e19.3 \u0026plusmn; 1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e46.2 \u0026plusmn; 2.5 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e30.2 \u0026nbsp;\u0026plusmn; 1.1 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003ePlasma IL-6 (ng/ml)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e1.16 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e98.5 \u0026plusmn; 8.4 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e41.8 \u0026plusmn; 5.08 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" style=\"width: 631px;\"\u003e\n \u003cp\u003eRenal Tissue Biomarkers\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eKidney SIRT-1 Level \u0026nbsp;(pg/mg protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e1.95 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e0.86 \u0026plusmn; 0.2 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e1.22 \u0026plusmn;\u0026nbsp;0.09 #\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 177px;\"\u003e\n \u003cp\u003eKidney Nrf-2 Level \u0026nbsp;(pg/mg protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e9.6 \u0026plusmn; 1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e3.07 \u0026plusmn; 0.2 **\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 151px;\"\u003e\n \u003cp\u003e7.4 \u0026plusmn; 1.1 ##\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbv. BUN: blood urea nitrogen; IL-6: interleukin-6; KIM-1: kidney injury molecule-1; MDA: malondialdehyde; NGAL: neutrophil gelatinase-associated lipocalin; Nrf2: nuclear factor erythroid 2\u0026ndash;related factor 2; SIRT-1: sirtuin-1; TNF-\u0026alpha;: tumor necrosis factor-alpha.\u003c/p\u003e\n\u003cp\u003eResults were presented as mean \u0026plusmn; SEM. *p\u0026lt;0.01, ** p\u0026lt;0.001 (different from control group); # p\u0026lt;0.05, ## p\u0026lt;0.001 (different from Colistin and tap water group).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this experimental study, we investigated the nephroprotective potential of wogonin in a rat model of CIN. Colistin, administered intraperitoneally at 20 mg/kg, successfully induced AKI, as reflected by elevated plasma levels of BUN, creatinine, KIM-1, NGAL, and MDA, along with pronounced tubular necrosis and inflammation. Wogonin, given at 50 mg/kg/day for 10 days with oral gavage, significantly mitigated these effects, reducing functional (BUN, creatinine) and injury markers (KIM-1, NGAL), attenuating oxidative stress and inflammatory mediators (MDA, TNF-α, IL-6), and restoring antioxidant tissue markers (SIRT-1, Nrf2). These effects were accompanied by notable histological improvement, including reductions in tubular epithelial necrosis, luminal debris, and tubular dilatation. To the best of our knowledge, this is the first report demonstrating the nephroprotective role of wogonin in a CIN model.\u003c/p\u003e\n\u003cp\u003eThe primary finding of this study was the significant histopathological improvement observed in the colistin + wogonin group, underscoring its potent nephroprotective effects against CIN. Notable reductions in tubular epithelial necrosis, luminal debris accumulation, and tubular dilatation were evident, indicating preservation of renal microarchitecture and suggesting attenuation of tissue-level injury. These histological improvements are consistent with previous studies in diabetic nephropathy, cisplatin-induced nephropathy and ureteral obstruction models, where wogonin attenuated renal injury through modulation of the PI3K/Akt/NF-κB signaling axis and suppression of fibrotic and apoptotic processes \u003csup\u003e9,10,12,15,21,22\u003c/sup\u003e. Furthermore, wogonin’s ability to inhibit the TGF-β/Smad3 pathway, an established mediator of renal fibrosis, has been demonstrated in diabetic nephropathy, reinforcing its antifibrotic potential \u003csup\u003e9\u003c/sup\u003e. Consistent with literature, the structural improvements observed in our histological findings, likely reflecting the antifibrotic properties of wogonin, corroborate the biochemical results and further emphasize the therapeutic potential of wogonin in maintaining renal integrity under nephrotoxic stress. Nevertheless, fibrotic remodeling was not specifically assessed using dedicated markers such as Masson's trichrome staining or alpha-smooth muscle actin (α-SMA) immunohistochemistry. Considering the known anti-fibrotic actions of wogonin, future studies should incorporate fibrosis-specific endpoints to comprehensively evaluate its long-term structural benefits.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElevated plasma levels of BUN and creatinine in the colistin + tap water group confirmed significant renal impairment and validated the successful establishment of the nephrotoxicity model. However, these conventional markers may not fully capture early or localized tubular injury. To overcome this limitation, we also measured plasma levels of KIM-1 and NGAL, biomarkers that are more sensitive to tubular epithelial damage. KIM-1 reflects proximal tubular injury, while NGAL is associated with both acute tubular damage and inflammatory responses \u003csup\u003e23,24\u003c/sup\u003e. The significant reductions in these biomarkers following wogonin treatment suggest substantial attenuation of renal injury at both functional and cellular levels. Meng et al. \u003csup\u003e12\u003c/sup\u003e reported that wogonin alleviated cisplatin-induced AKI by suppressing pro-inflammatory cytokines and oxidative stress, leading to decreased serum BUN, creatinine and KIM-1 levels. Similarly, Badawy et al. \u003csup\u003e14\u003c/sup\u003e demonstrated that wogonin reduced serum BUN, creatinine by inhibiting NF-κB and MAPK pathways in cisplatin-induced AKI models. In nephrotoxicity models in the literature, the nephroprotective effects of wogonin have generally been evaluated using only traditional functional biomarkers. Thus, the inclusion and modulation of early-response injury biomarkers such as NGAL and KIM-1, as demonstrated in our study, provide an important contribution to the literature by enabling more sensitive detection and evaluation of renal injury.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith previous similar nephrotoxicity patterns, CIN represents a well-characterized and clinically significant form of drug-induced renal injury, primarily mediated through oxidative stress, inflammation, and apoptosis \u003csup\u003e2,3,5,8,25\u003c/sup\u003e. In the present study, significant elevations in plasma TNF-α, IL-6, and MDA levels following colistin exposure reflect the activation of systemic inflammatory and oxidative stress pathways. Wogonin treatment effectively attenuated these aberrations, indicating its potential role in modulating redox homeostasis and inflammatory signaling. These effects are supported by earlier experimental studies indicating that wogonin exerts anti-inflammatory and antioxidant activity by suppressing nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling cascades \u003csup\u003e9,10,13,15\u003c/sup\u003e. Although no prior studies have investigated wogonin in a CIN model, Badawy et al. \u003csup\u003e14\u003c/sup\u003e demonstrated in a cisplatin-induced nephrotoxicity model that wogonin pretreatment significantly attenuated renal expression of IL-1β, TNF-α, and NF-κB, emphasizing its potent anti-inflammatory properties in renal injury settings. Similarly, Zheng et al. \u003csup\u003e9\u003c/sup\u003e reported that wogonin attenuated renal inflammation and fibrosis in a diabetic nephropathy model by reducing proinflammatory cytokines, including TNF-α and IL-1β, through inhibition of the NF-κB and TGF-β1/Smad3 signaling pathways. Mechanistically, wogonin has been shown to inhibit the nuclear translocation of NF-κB and suppress the expression of inducible nitric oxide synthase (iNOS), both of which are important mediators in the propagation of proinflammatory signaling pathways \u003csup\u003e26,27\u003c/sup\u003e. These molecular actions may underlie the nephroprotective effects observed in our study. However, while our findings support the functional and histopathological efficacy of wogonin, they are limited by the absence of direct molecular analyses of inflammatory and apoptotic pathways. This gap restricts the mechanistic resolution of the observed effects. Future investigations should incorporate pathway-specific analyses, including necroptosis, autophagy, and apoptosis-related signaling, to elucidate the precise molecular pathways of wogonin’s nephroprotective activity in CIN models.\u003c/p\u003e\n\u003cp\u003eSIRT-1 and Nrf2 are substantial regulators of cellular stress responses, apoptosis, and antioxidant defense mechanisms. In the context of AKI, both molecules serve as critical modulators of renal cell survival and protection against oxidative damage \u003csup\u003e28–30\u003c/sup\u003e. In our study, wogonin administration resulted in a significant upregulation of SIRT-1 and Nrf2 expression in renal tissue, indicating activation of endogenous cytoprotective pathways. The restoration of SIRT-1 expression is particularly relevant, given its known role in promoting mitochondrial function, regulating apoptotic signaling, and attenuating inflammation \u003csup\u003e28,29\u003c/sup\u003e. Previous studies have demonstrated that wogonin upregulates Nrf2 expression in various models of tissue injury, including acute lung injury, cardiac injury, hepatic damage, ischemia-reperfusion injury, traumatic brain injury, and cancer \u003csup\u003e27,31–39\u003c/sup\u003e. This upregulation has been shown to inhibit pro-apoptotic Bcl-2-associated X protein (Bax) signaling and enhance B-cell lymphoma 2 (Bcl-2) mediated autophagy, thereby promoting cellular survival under oxidative stress \u003csup\u003e40\u003c/sup\u003e. Also, Nrf2 activation by wogonin supports its antioxidative capacity through the induction of downstream effectors such as heme oxygenase-1 (HO-1), thereby enhancing the renal antioxidant defense system and neutralizing reactive oxygen species \u003csup\u003e27,30,32\u003c/sup\u003e. In this context, our findings provide a valuable contribution to the existing literature, as one of the few studies to suggest that the protective renal effects of wogonin in a nephrotoxicity model. The concurrent upregulation of SIRT-1 and Nrf2 observed in our study implies a synergistic mechanism by which wogonin alleviates oxidative stress, inhibits apoptotic signaling, and supports the preservation of tubular epithelial integrity. However, despite demonstrating increased expression of these regulators, our study did not include direct evaluation of downstream effectors such as HO-1, the Bax/Bcl-2 ratio, or cleaved caspase-3, critical mediators in oxidative stress and apoptosis signaling. To clarify the mechanistic underpinnings of wogonin's nephroprotective effects in the context of CIN, future investigations should incorporate molecular approaches such as Western blotting or immunohistochemical analysis to characterize the downstream signaling events associated with SIRT-1 and Nrf2 activation.\u003c/p\u003e\n\u003cp\u003eThis study presents several notable strengths. It is among the first to comprehensively evaluate the nephroprotective potential of wogonin in a well-established model of CIN, integrating systemic and renal-specific biomarkers with rigorous histopathological assessment. The experimental design included controlled conditions, blinding during histological scoring, and the inclusion of both functional and injury-related markers, enhancing the validity and reproducibility of the findings. Importantly, the observed histological protection, characterized by reduced tubular necrosis and architectural preservation, provides structural corroboration of the biochemical improvements, reinforcing the translational relevance of wogonin in clinical scenarios where colistin use is necessary but nephrotoxicity is a limiting factor. Nevertheless, several limitations must be acknowledged. The study did not include direct measurements of glomerular filtration rate (GFR), which could have offered a more precise functional assessment. This omission is primarily due to the technical challenges associated with urine collection in rodents. Additionally, the investigation was limited to a single dosing regimen (50 mg/kg/day), selected based on prior literature. However, the absence of a dose–response analysis limits our ability to determine the optimal therapeutic window or identify potential toxicity thresholds. Moreover, although significant phenotypic improvements were documented, the study did not incorporate molecular analyses to elucidate the precise signaling pathways involved in wogonin’s mechanism of action. Future studies should aim to overcome these limitations by incorporating molecular tools to investigate necroptosis, autophagy, apoptosis, and fibrosis-related signaling. Dose–response studies and exploration of synergistic effects with existing nephroprotective agents across diverse nephrotoxicity models will further clarify the translational potential of wogonin as an adjunctive therapeutic strategy.\u003c/p\u003e\n\n"},{"header":"Conclusion","content":"\u003cp\u003eWogonin demonstrated significant nephroprotective efficacy in a rat model of CIN through the attenuation of oxidative stress, suppression of pro-inflammatory cytokine expression, and amelioration of tubular epithelial damage. These effects were characterized by histopathological improvements, and reductions in inflammatory and oxidative biomarkers, such as MDA, TNF-α, and IL-6, accompanied by the upregulation of key cytoprotective mediators, including SIRT-1 and Nrf2. These findings underscore the need for clinical evaluation of wogonin as a promising adjunctive therapy to protect renal function in contexts necessitating colistin use.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAKI: Acute kidney injury\u003c/p\u003e\n\u003cp\u003eAkt: Protein Kinase B\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-SMA: Alpha-smooth muscle actin\u003c/p\u003e\n\u003cp\u003eANOVA: One-way analysis of variance\u003c/p\u003e\n\u003cp\u003eBax: Bcl-2-associated X protein\u003c/p\u003e\n\u003cp\u003eBcl2: B-cell lymphoma 2\u003c/p\u003e\n\u003cp\u003eBUN: Blood urea nitrogen\u003c/p\u003e\n\u003cp\u003eCIN: Colistin-induced nephrotoxicity\u003c/p\u003e\n\u003cp\u003eELISA: Enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eGFR: Glomerular filtration rate\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E: Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eHO-1: Heme oxygenase-1\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta;: Interleukin-1 beta\u003c/p\u003e\n\u003cp\u003eIL-6: Interleukin-6\u003c/p\u003e\n\u003cp\u003eiNOS: Inducible nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eKIM-1: Kidney injury molecule-1\u003c/p\u003e\n\u003cp\u003eMAPK: Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003eMDA: Malondialdehyde\u003c/p\u003e\n\u003cp\u003eNF-\u0026kappa;B: Nuclear factor-kappa B\u003c/p\u003e\n\u003cp\u003eNGAL: Neutrophil gelatinase-associated lipocalin\u003c/p\u003e\n\u003cp\u003enM: Nanomoles\u003c/p\u003e\n\u003cp\u003eNrf2: Nuclear factor erythroid 2\u0026ndash;related factor 2\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePI3K: Phosphatidylinositol 3-Kinase\u003c/p\u003e\n\u003cp\u003eSEM: Standard error of the mean\u003c/p\u003e\n\u003cp\u003eSIRT-1: Sirtuin-1\u003c/p\u003e\n\u003cp\u003eSmad3: Mothers against decapentaplegic homolog 3\u003c/p\u003e\n\u003cp\u003eTBARS: Thiobarbituric acid reactive substances\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;: Transforming Growth Factor-beta\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha;: Tumor necrosis factor-alpha\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.S.T., M.A.E. and O.E. performed the animal experiments, including group allocation, treatment administration, and sample collection. A.S.T., B.E.G. and M.A.C assisted in experimental design, manuscript revision, and contributed to the interpretation of results. M.A.E. and O.E. conducted the biochemical analyses and helped prepare figures and tables. The authors confirm that no paper mill and artificial intelligence was used. All authors contributed to the study design, read, and approved the final manuscript, and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to institutional privacy policies and ethical considerations, the datasets generated and analyzed during the current study are not publicly available but can be made available upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Animal Care and Ethical Committee of the Science University, Istanbul (Ethics Approval Number: 2925042821). All procedures involving animals were conducted in full compliance with the internationally recognized ARRIVE 2.0 guidelines and were designed to adhere strictly to the principles of replacement, reduction, and refinement (the 3Rs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict and Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest related to this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTsuji, B. T. et al. International Consensus Guidelines for the Optimal Use of the Polymyxins: Endorsed by ACCP, ESCMID, IDSA, ISAP, SCCM, and SIDP. Pharmacotherapy. 39, 10\u0026ndash;39 (2019).\u003c/li\u003e\n\u003cli\u003eChien, H. T., Lin, Y. C., Sheu, C. C., Hsieh, K. P. \u0026amp; Chang, J. S. Is colistin-associated acute kidney injury clinically important in adults? A systematic review and meta-analysis. Int. J. Antimicrob. Agents. 55, 105889 (2020).\u003c/li\u003e\n\u003cli\u003eGai, Z., Samodelov, S. L., Kullak-Ublick, G. A. \u0026amp; Visentin, M. Molecular mechanisms of colistin-induced nephrotoxicity. Molecules. 24, 653 (2019).\u003c/li\u003e\n\u003cli\u003eFalagas, M. E. et al. Clinical use of intravenous polymyxin B for multidrug-resistant Gram-negative infections: An evaluation of the current evidence. J. Glob. Antimicrob. Resist. 24, 342\u0026ndash;359 (2021).\u003c/li\u003e\n\u003cli\u003eOrdooei Javan, A., Shokouhi, S. \u0026amp; Sahraei, Z. A review on colistin nephrotoxicity. Eur. J. Clin. Pharmacol. 71, 801\u0026ndash;810 (2015).\u003c/li\u003e\n\u003cli\u003eBintang, M. A. K. M., Nopparat, J. \u0026amp; Srichana, T. In vivo evaluation of nephrotoxicity and neurotoxicity of colistin formulated with sodium deoxycholate sulfate in mice. Naunyn Schmiedebergs Arch. Pharmacol. 396, 3243\u0026ndash;3252 (2023).\u003c/li\u003e\n\u003cli\u003eJafari, F. \u0026amp; Elyasi, S. Prevention of colistin-induced nephrotoxicity: a review of preclinical and clinical data. Expert Rev. Clin. Pharmacol. 14, 1113\u0026ndash;1131 (2021).\u003c/li\u003e\n\u003cli\u003eHeybeli, C., Oktan, M. A. \u0026amp; \u0026Ccedil;avdar, Z. Rat models of colistin nephrotoxicity: previous experimental research and future perspectives. Eur. J. Clin. Microbiol. Infect. Dis. 38, 1387\u0026ndash;1393 (2019).\u003c/li\u003e\n\u003cli\u003eZheng, Z. C. et al. Wogonin ameliorates renal inflammation and fibrosis by inhibiting NF-\u0026kappa;B and TGF-\u0026beta;1/Smad3 pathways in diabetic nephropathy. Drug Des. Devel. Ther. 14, 4135\u0026ndash;4148 (2020).\u003c/li\u003e\n\u003cli\u003eLei, L. et al. Wogonin alleviates kidney tubular epithelial injury in diabetic nephropathy by inhibiting PI3K/Akt/NF-\u0026kappa;B signaling. Drug Des. Devel. Ther. 15, 3131\u0026ndash;3150 (2021).\u003c/li\u003e\n\u003cli\u003eTalbi, A. et al. Pharmacokinetics, tissue distribution, excretion and plasma protein binding studies of wogonin in rats. Molecules. 19, 5538\u0026ndash;5549 (2014).\u003c/li\u003e\n\u003cli\u003eMeng, X. M. et al. Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis. Lab Invest. 98, 79\u0026ndash;94 (2018).\u003c/li\u003e\n\u003cli\u003eLiu, Y. et al. Wogonin upregulates SOCS3 to alleviate injury in diabetic nephropathy by inhibiting TLR4-mediated JAK/STAT/AIM2 signaling. Mol. Med. 30, 78 (2024).\u003c/li\u003e\n\u003cli\u003eBadawy, A. M. et al. Wogonin pre-treatment attenuates cisplatin-induced nephrotoxicity in rats: Impact on PPAR-\u0026gamma;, inflammation, apoptosis, and Wnt/\u0026beta;-catenin. Chem. Biol. Interact. 308, 137\u0026ndash;146 (2019).\u003c/li\u003e\n\u003cli\u003eKuo, H. L. et al. Wogonin ameliorates ER stress-associated inflammation, apoptosis and fibrosis in unilateral ureteral obstruction in mice. Eur. J. Pharmacol. 977, 176676 (2024).\u003c/li\u003e\n\u003cli\u003ePercie du Sert, N. et al. Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. 18, e3000411 (2020).\u003c/li\u003e\n\u003cli\u003eTannenbaum, J. \u0026amp; Bennett, B. T. Russell and Burch\u0026rsquo;s 3Rs then and now: The need for clarity in definition and purpose. J. Am. Assoc. Lab. Anim. Sci. 54, 120 (2005).\u003c/li\u003e\n\u003cli\u003eSivanesan, S. S. et al. Gelofusine ameliorates colistin-induced nephrotoxicity. Antimicrob. Agents Chemother. 61, e00985-17 (2017).\u003c/li\u003e\n\u003cli\u003eErbas, O. et al. Oxytocin alleviates cisplatin-induced renal damage in rats. Iran. J. Basic Med. Sci. 17, 747\u0026ndash;752 (2014).\u003c/li\u003e\n\u003cli\u003eFaul, F., Erdfelder, E., Lang, A. G. \u0026amp; Buchner, A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods. 39, 175\u0026ndash;191 (2007).\u003c/li\u003e\n\u003cli\u003eWang, Y. et al. Wogonin induces apoptosis and reverses sunitinib resistance in renal carcinoma via CDK4-RB inhibition. Front. Pharmacol. 11, 1152 (2020).\u003c/li\u003e\n\u003cli\u003eLi, J. et al. Impact of baicalin and Scutellaria baicalensis components on renal fibrosis in diabetic kidney disease. Front. Pharmacol. 15, 1480626 (2024).\u003c/li\u003e\n\u003cli\u003eVaidya, V. S. et al. Kidney injury molecule-1 outperforms traditional kidney biomarkers in preclinical studies. Nat. Biotechnol. 28, 478\u0026ndash;485 (2010).\u003c/li\u003e\n\u003cli\u003eHaase, M. et al. Accuracy of NGAL in diagnosis and prognosis of acute kidney injury: A meta-analysis. Am. J. Kidney Dis. 54, 1012\u0026ndash;1024 (2009).\u003c/li\u003e\n\u003cli\u003eDai, C. et al. Colistin-induced nephrotoxicity involves mitochondrial, death receptor, and ER pathways in mice. Antimicrob. Agents Chemother. 58, 4075\u0026ndash;4085 (2014).\u003c/li\u003e\n\u003cli\u003eYao, J. et al. Wogonin prevents LPS-induced acute lung injury via PPAR\u0026gamma;-mediated NF-\u0026kappa;B inhibition. Immunology. 143, 241\u0026ndash;257 (2014).\u003c/li\u003e\n\u003cli\u003eHe, X. et al. Wogonin attenuates inflammation and oxidative stress in mastitis by Akt/NF-\u0026kappa;B inhibition and Nrf2/HO-1 activation. Cell Stress Chaperones. 28, 989\u0026ndash;999 (2023).\u003c/li\u003e\n\u003cli\u003eAlbalawi, R. S. et al. Parthenolide phytosomes attenuate gentamicin-induced nephrotoxicity via SIRT-1, Nrf2, HO-1, and NQO1. Molecules. 28, 2741 (2023).\u003c/li\u003e\n\u003cli\u003eKumari, A., Sodum, N., Ravichandiran, V. \u0026amp; Kumar, N. Role of SIRT-1 in treatment and prevention of diabetic nephropathy: A review. Curr. Mol. Pharmacol. 16, 811\u0026ndash;831 (2023).\u003c/li\u003e\n\u003cli\u003eZaghloul, R. A., Abdelghany, A. M. \u0026amp; Samra, Y. A. Rutin and selenium nanoparticles protect against diabetic nephropathy by modulating Jak-2/Stat3 and Nrf2/HO-1. Eur. J. Pharmacol. 933, 175289 (2022).\u003c/li\u003e\n\u003cli\u003eZhou, Y., Dou, F., Song, H. \u0026amp; Liu, T. Wogonin exerts anti-ulcerative effects in colitis via Nrf2/TLR4/NF-\u0026kappa;B pathway. Environ. Toxicol. 37, 954\u0026ndash;963 (2022).\u003c/li\u003e\n\u003cli\u003eFeng, Y. et al. Wogonin protects against brain injury by reducing oxidative stress via PI3K/Nrf2/HO‑1 pathway. Int. J. Mol. Med. 49, 53 (2022).\u003c/li\u003e\n\u003cli\u003eLiu, X. et al. Wogonin induces ferroptosis in pancreatic cancer via Nrf2/GPX4 axis inhibition. Front. Pharmacol. 14, 1129662 (2023).\u003c/li\u003e\n\u003cli\u003eDai, J. M. et al. Wogonin alleviates liver injury in sepsis through Nrf2-mediated NF-\u0026kappa;B suppression. J. Cell. Mol. Med. 25, 5782\u0026ndash;5798 (2021).\u003c/li\u003e\n\u003cli\u003eYang, H. et al. Wogonin attenuates inflammation and oxidative stress in acute lung injury via PPAR\u0026alpha;, AKT, and NRF2 pathways. Naunyn Schmiedebergs Arch. Pharmacol. (2025).\u003c/li\u003e\n\u003cli\u003eCheng, Z. et al. Wogonin alleviates NLRP3 inflammasome activation after cerebral ischemia-reperfusion via AMPK/SIRT1. Brain Res. Bull. 207, 110886 (2024).\u003c/li\u003e\n\u003cli\u003eGe, J., Yang, H., Yu, N., Lin, S. \u0026amp; Zeng, Y. Wogonin alleviates sepsis-induced lung injury by modulating macrophage polarization via SIRT1\u0026ndash;FOXO1. Tissue Cell. 88, 102400 (2024).\u003c/li\u003e\n\u003cli\u003eWang, L. et al. Cardiac protection by wogonin in pulmonary fibrosis via Sirt1/\u0026gamma;-H2AX pathway. Front. Pharmacol. 16, 1551141 (2025).\u003c/li\u003e\n\u003cli\u003eQian, C. et al. Wogonin enhances ROS-induced apoptosis and potentiates chemo effects via Nrf2 suppression in HepG2 cells. Free Radic. Res. 48, 607\u0026ndash;621 (2014).\u003c/li\u003e\n\u003cli\u003eLiu, X. Q. et al. Wogonin protects podocytes via Bcl-2-mediated autophagy and apoptosis inhibition in diabetic kidney disease. Acta Pharmacol. Sin. 43, 96\u0026ndash;110 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Colistin, colistin-induced nephrotoxicity, inflammation, nephroprotection, oxidative stress, Scutellaria baicalensis, wogonin","lastPublishedDoi":"10.21203/rs.3.rs-7124382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7124382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Colistin remains a critical antibiotic for treating multidrug-resistant Gram-negative infections, yet its clinical use is severely limited by its high nephrotoxicity profile. There is an urgent need for nephroprotective agents that can mitigate colistin-induced nephrotoxicity (CIN) without compromising antimicrobial efficacy. Wogonin, a flavonoid compound with known antioxidant and anti-inflammatory properties, has shown promise in various models of renal injury, though its potential role in CIN has not been previously evaluated. This study aimed to assess the nephroprotective efficacy of wogonin in a well-characterized Wistar rat model of CIN, using a combination of biochemical, molecular, and histopathological parameters. Thirty adult female Wistar rats were randomly assigned into three groups: control, colistin + tap water, and colistin + wogonin (50 mg/kg/day, oral gavage for 10 days). CIN was induced using a single intraperitoneal dose of colistin (20 mg/kg). Plasma levels of blood urea nitrogen (BUN), creatinine, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were measured, along with renal tissue levels of SIRT-1 and Nrf2. Histopathological evaluation was performed using a semi-quantitative scoring system. Colistin administration resulted in significant renal dysfunction and tubular injury, as evidenced by elevated biochemical markers and histological damage. Wogonin treatment significantly attenuated elevations in BUN, creatinine, KIM-1, NGAL, MDA, IL-6, and TNF-α, while restoring sirtuin-1 (SIRT-1) and nuclear factor erythroid 2–related factor 2 (Nrf2) levels. Histopathological analysis revealed reduced tubular necrosis, luminal debris, and dilatation in the wogonin group. Wogonin demonstrated significant nephroprotective effects in a rat model of CIN by reducing oxidative stress, suppressing pro-inflammatory cytokine expression, and mitigating tubular epithelial injury.","manuscriptTitle":"Protective Effect of Wogonin Against Colistin-Induced Nephrotoxicity in Wistar Rats: A Controlled Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 17:48:38","doi":"10.21203/rs.3.rs-7124382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-11T15:53:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T22:22:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"276362631124354785927054004207936553653","date":"2026-02-19T16:54:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T07:15:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"228446489408039248737232531415076032570","date":"2025-10-20T01:30:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T08:51:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-08T06:54:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-18T12:08:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-18T11:52:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ebf7624-87dc-4925-87dd-4bbfb4884a6c","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53379781,"name":"Biological sciences/Biochemistry"},{"id":53379782,"name":"Health sciences/Diseases"},{"id":53379783,"name":"Biological sciences/Drug discovery"},{"id":53379784,"name":"Health sciences/Medical research"},{"id":53379785,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-05-20T09:39:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 17:48:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7124382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7124382","identity":"rs-7124382","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}