Amelioration of Doxorubicin-Mediated Nephrotoxicity through Antioxidant and Anti-apoptotic Mechanisms of 5,4′-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone

Amelioration of Doxorubicin-Mediated Nephrotoxicity through Antioxidant and Anti-apoptotic Mechanisms of 5,4′-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone | 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 Research Article Amelioration of Doxorubicin-Mediated Nephrotoxicity through Antioxidant and Anti-apoptotic Mechanisms of 5,4′-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone Peramaiyan Rajendran, Abdullah Alzahrani, Ramya Sekar, Gamal M Bekhet, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8027411/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted 9 You are reading this latest preprint version Abstract Doxorubicin (DOX) is a potent chemotherapeutic agent whose clinical utility is limited by cumulative nephrotoxicity driven by oxidative stress, inflammation, and apoptosis. Natural flavonoids have shown promise in mitigating such adverse effects. This study evaluated the renoprotective efficacy of 5,4′-dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone (DDR) against chronic DOX-induced kidney injury, focusing on the involvement of NOX-4-mediated oxidative stress, inflammatory signalling, NRF2 antioxidant pathways, and apoptotic regulators BCL2 and Caspase-3.Male albino mice were randomized into five groups (n = 6): control, DOX (2.5 mg/kg intraperitoneally, once weekly for six weeks), DOX plus low-dose DDR (25 mg/kg, twice weekly), DOX plus high-dose DDR (50 mg/kg, twice weekly), and DDR alone. Renal function was assessed via serum creatinine, urea, and biomarkers NGAL and KIM-1. Oxidative stress markers (MDA, GSH, SOD, CAT), pro-inflammatory cytokines (pNFκB,TNF-α, IL-6 and IL-1β), and protein expression of NOX-4, NRF2, BCL2, and Caspase-3 were quantified by biochemical assays, ELISA, immunohistochemistry, and Western blotting. Kidney tissues underwent histopathological evaluation using hematoxylin and eosin and Masson’s trichrome staining. DOX administration induced significant renal impairment, characterized by elevated MDA, NOX-4, pNFκB,TNF-α, IL-6, Il-1β and Caspase-3 levels, alongside reduced antioxidant enzyme activities and BCL2 expression. Activation of signaling and suppression of NRF2 correlated with marked glomerular and tubular injury. DDR treatment ameliorated these effects in a dose-dependent manner; high-dose DDR significantly improved renal function, diminished oxidative and inflammatory mediators, enhanced antioxidant defenses, suppressed NOX-4 and upregulated NRF2 and BCL2, resulting in preserved renal histology. DDR confers dose-dependent nephroprotection against chronic DOX-induced toxicity by modulating oxidative stress, inflammation, and apoptosis. These findings advocate DDR’s potential as an adjunctive agent to alleviate chemotherapy-associated renal damage. Doxorubicin NOX-4 NFκB nephrotoxicity flavonoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Doxorubicin (DOX) is a widely used anthracycline chemotherapy drug, effective against a range of cancers including breast cancer, lymphomas, and sarcomas (Almajidi et al., 2023 ; Sandal et al., 2023 ). Despite its clinical success, the use of DOX is often curtailed by its cumulative toxicity across various organs. While cardiac toxicity is well-documented, nephrotoxicity has increasingly been recognized as a critical limitation, particularly with extended treatment regimens (Al-Ali et al., 2024; Espírito Santo et al., 2023 ). Due to their rich blood supply and high metabolic activity, the kidneys are especially vulnerable to damage induced by DOX, primarily related to oxidative stress and inflammation. The mechanism of DOX-induced kidney injury involves overproduction of reactive oxygen species (ROS), largely mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, especially NOX-4 (Zhang et al., 2024a; Zheng et al., 2022 ). This oxidative imbalance triggers lipid and protein damage and disrupts mitochondrial function. Concurrently, DOX promotes the release of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which drive renal inflammation (Entezari Heravi et al., 2018). Activation of the inflammatory and apoptotic responses, while suppression of the antioxidant defense pathway governed by nuclear factor erythroid 2–related factor 2 (NRF2) weakens cellular resilience (Maashi et al., 2022). Collectively, these pathogenic processes result in damage to glomerular and tubular structures, interstitial fibrosis, and progressive loss of renal function. Naturally occurring bioactive molecules, particularly flavonoids, have attracted considerable attention for their diverse protective effects against drug-induced organ toxicity (Rahmani et al., 2023 ; Singh et al., 2015 ). These compounds possess strong antioxidant, anti-inflammatory, and anti-apoptotic properties, making them promising agents for kidney protection (Al-Khayri et al., 2022 ; Chagas et al., 2022 ; Zhang et al., 2024b ). Among them, 5,4′-dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone (DDR), a methoxylated flavonoid glycoside, has shown noteworthy pharmacological actions (Al-Saeedi and Rajendran, 2024 ; AlZahrani et al., 2024 ). DDR’s ability to regulate oxidative balance, modulate inflammatory mediators, and influence apoptotic pathways supports its potential in mitigating DOX-associated renal injury. This study aimed to explore the protective effects of DDR in a mouse model of chronic DOX-induced nephrotoxicity, focusing on its impact on oxidative stress, inflammation, apoptosis, and key signaling pathways including NOX-4, and NRF2. To our knowledge, this investigation provides a comprehensive analysis of the molecular mechanisms underlying DDR’s renoprotective effects against DOX toxicity. 2 Materials and Methods 2.1 Chemicals Doxorubicin hydrochloride (analytical grade) was purchased from Sigma-Aldrich (USA). Serum urea and creatinine assay kits were obtained from BioMed and Spectrum Diagnostics (Cairo, Egypt). ELISA kits for kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were sourced from Elabscience and CUSABIO (USA). Assay kits for malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were procured from Cayman Chemical (USA). ELISA kits for tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), along with primary antibodies against TNF-α, IL-6, nuclear factor erythroid 2-related factor 2 (NRF2), B-cell lymphoma 2 (BCL2), and Caspase-3 for Western blotting, were supplied by Thermo Fisher Scientific (USA). Chromogenic substrates for immunohistochemistry (DAB), hematoxylin and eosin (H&E), and Masson’s trichrome staining reagents were purchased from standard histology suppliers. All other reagents used were of analytical grade. 2.2 Isolation of DDR 5,4′-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone (DDR) was isolated from the aerial parts of Indigofera aspalathoides Vahl. Plant authentication and extraction procedures followed previously established protocols (Al-Saeedi and Rajendran, 2024 ; AlZahrani et al., 2024 ). Briefly, dried powdered plant material was subjected to methanol extraction, followed by sequential solvent fractionation. The flavonoid-rich fraction was purified via silica gel column chromatography and preparative high-performance liquid chromatography (HPLC), yielding DDR with a purity exceeding 98%, confirmed by HPLC and nuclear magnetic resonance (NMR) spectroscopy. The purified compound was stored at − 20°C in amber vials until use. 2.3 Experimental Animals Male Swiss albino mice (6–7 weeks old, weighing 23 ± 3 g) were obtained from the Experimental Surgery & Animal Laboratory, King Saud University, Riyadh, Saudi Arabia. Animals were acclimated for one week prior to experimentation under standard conditions: temperature 25 ± 2°C, relative humidity 60–70%, and a 12-hour light/dark cycle. Animal care and use complied with the Guide for the Care and Use of Laboratory Animals (NIH) and the ARRIVE guidelines. They were housed in polypropylene cages with ad libitum access to commercial pellet diet and water. All procedures complied with institutional ethical guidelines approved by the Research Ethics Committee, King Faisal University, Hofuf, Saudi Arabia (Approval No. ETHICS3529). 2.4 Experimental Design Following acclimatization, mice were randomly assigned to five groups (n = 6/group): Group I (Control): Received normal saline and vehicle (≤ 5% DMSO in 0.5% CMC-Na) intraperitoneally for six weeks. Group II (DOX): Received DOX 2.5 mg/kg intraperitoneally, once weekly for six weeks. Group III (DOX + DDR Low Dose): Received DOX as above plus DDR 25 mg/kg intraperitoneally, twice weekly for six weeks. Group IV (DOX + DDR High Dose): Received DOX as above plus DDR 50 mg/kg intraperitoneally, twice weekly for six weeks. Group V (DDR Alone): Received DDR 50 mg/kg intraperitoneally, twice weekly for six weeks. 2.5 Sample Collection At the end of the treatment period, mice were anesthetized with isoflurane 4%. Blood was collected by cervical decapitation, and serum was separated by centrifugation at 4000 rpm for 15 minutes. Serum samples were used for renal function tests. Kidneys were excised, rinsed with saline, and blotted dry. One kidney from each mouse was fixed in 10% neutral buffered formalin for histological and immunohistochemical analysis, while the contralateral kidney was snap-frozen at − 80°C for biochemical and Western blot assays. 2.6 Renal Function Assessment Serum creatinine and urea levels were measured using enzymatic colorimetric kits following the manufacturers’ protocols. Absorbance was recorded using a microplate reader, and concentrations were determined against calibration curves. 2.7 Measurement of Inflammatory and Renal Injury Biomarkers Serum levels of IL-6, TNF-α, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) were quantified using specific ELISA kits. Standards and samples were assayed in duplicates, measuring absorbance at 450 nm with 570 nm reference wavelength. 2.8 Determination of Renal Oxidative Stress Markers Kidney tissues were homogenized in ice-cold 10 mM potassium phosphate buffer (pH 7.4) at a 5:1 buffer-to-tissue ratio and centrifuged at 4°C for 10 minutes. The supernatant was used to measure malondialdehyde (MDA) concentration via thiobarbituric acid reactive substances (TBARS) assay, and activities of reduced glutathione (GSH) and superoxide dismutase (SOD) were determined by established biochemical methods. 2.9 Quantification of NOX-4 and Prostaglandin E2 (PGE-2) Renal homogenates were assessed for NADPH oxidase 4 (NOX-4) and prostaglandin E2 (PGE-2) concentrations using mouse-specific ELISA kits, conducted per the manufacturers’ instructions. 2.10 Western Blot Analysis Kidney tissues were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors, followed by centrifugation at 12,000 × g for 15 minutes at 4°C. Protein concentrations were determined using BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked and incubated overnight at 4°C with primary antibodies targeting pNFκB, NFκB, TNF-α, IL-6, IL-1β, NRF2, BCL2, and Caspase-3. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using enhanced chemiluminescence. Densitometric analysis was performed with ImageJ software, normalizing protein levels to β-actin controls. 2.11 Histopathological Examination Formalin-fixed kidneys were processed, paraffin-embedded, sectioned at 5 µm thickness, and stained with hematoxylin and eosin (H&E) for general morphology. 2.11 Immunohistochemistry Renal sections were deparaffinized, rehydrated, and endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. Non-specific binding was blocked with normal goat serum. Sections were incubated overnight at 4°C with anti-TNF-α antibody (1:100 dilution), followed by incubation with HRP-conjugated secondary antibody. The antigen-antibody complexes were visualized using DAB substrate. 2.12 Masson’s Trichrome Staining Kidney sections (5 µm) were stained with Masson’s trichrome to assess collagen deposition and fibrosis. The staining protocol included sequential incubation with Weigert’s iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue. Stained sections were dehydrated, cleared, and mounted. Collagen content was quantified by calculating the percentage of blue-stained area using ImageJ software. 2.13 Statistical Analysis Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism software. Differences were considered statistically significant at p < 0.05. 3 Results 3.1 Effect of DDR on kidney function The chemical structure of DDR (Fig. 1 A) and experimental design (Fig. 1 B) are shown. DOX administration significantly elevated serum KIM-1, creatinine, urea, and NGAL compared with controls (Figs. 1 C–F). DOX markedly elevated serum KIM-1 compared with controls, while DDR reduced levels dose-dependently, with the high dose restoring near baseline. Serum creatinine increased robustly in DOX mice, which DDR significantly attenuated, particularly at high dose. Urea followed a similar pattern, with DDR lowering levels close to controls. NGAL was also dramatically increased in DOX mice, but DDR normalized values in a dose-dependent manner. DDR alone did not differ from controls. DDR supplementation reduced all markers in a dose-dependent manner, with high-dose DDR restoring values close to baseline. DDR alone had no adverse effect, confirming its safety. 3.2 Effect of DDR on inflammatory cytokines DOX markedly increased circulating IL-6 and TNF-α (Figs. 2 A, B). IL-6 levels varied significantly among groups. DOX induced a robust increase, while DDR significantly reduced IL-6 in both low- and high-dose groups, with the latter restoring levels to near baseline. A similar trend was observed for TNF-α, with high-dose DDR providing the greatest suppression. DDR significantly suppressed these elevations, with the high dose normalizing cytokine levels. DDR alone was indistinguishable from controls. 3.3 Renal Expression of TNF-α and IL-6 Western blot (Figs. 2 C and D) and immunohistochemistry TNF-α (Fig. 3 ) confirmed strong upregulation of TNF-α and IL-6 in DOX-treated kidneys. DDR reduced both proteins dose-dependently, with high-dose treatment restoring near-control levels. Western blot analysis confirmed marked upregulation of TNF-α in DOX-treated kidneys. DDR supplementation significantly and dose-dependently reduced expression, with high-dose treatment restoring values close to baseline. Immunohistochemistry supported these findings: DOX kidneys exhibited group showing positive immunostaining of the glomerulus (red arrow) and tubules (block arrow), whereas DDR reduced staining in a dose-dependent manner. DDR alone showed no difference from controls. 3.4 Effect of DDR on oxidative stress DOX induced severe oxidative stress, reflected by increased MDA, PGE₂, and NOX-4, and depletion of catalase, GSH, and SOD (Figs. 4 A–F). DOX significantly increased renal MDA, and NOX-4 while reducing catalase, GSH, and SOD. DDR supplementation significantly reversed these alterations in a dose-dependent fashion, with high-dose DDR restoring all parameters near baseline. DDR alone remained comparable to controls. 3.5 Effect of DDR on NRF2/HO-1/NQO-1 Pathway DOX markedly suppressed NRF2, HO-1, and NQO-1 expression (Figs. 5 A, B). DOX profoundly suppressed NRF2, and NQO-1. DDR treatment restored expression in a dose-dependent manner, with high-dose DDR normalizing all three proteins. DDR alone slightly elevated HO-1 above baseline but otherwise showed no deviation from controls. 3.6 Histopathological Alterations Control kidneys displayed intact glomeruli and tubules (Fig. 6 ). DOX caused severe pathology, including lymphocytic infiltration, hemorrhage, vacuolization, necrosis, tubular dilation, and epithelial atrophy. DDR afforded dose-dependent protection: low-dose DDR partially ameliorated injury, while high-dose DDR preserved architecture with minimal inflammation and necrosis. DDR alone maintained normal histology, verifying its safety. 3.7 Effect of DDR on renal fibrosis Masson’s trichrome staining revealed extensive interstitial fibrosis, perivascular thickening, and collagen deposition in DOX kidneys (Fig. 7 ). DDR supplementation reduced collagen deposition in a dose-dependent manner, with high-dose DDR showing marked antifibrotic protection. 3.9 Apoptotic Markers DOX decreased anti-apoptotic BCL2 and BCL-XL while elevating cleaved caspase-3 (Figs. 8 A, B). DOX suppressed anti-apoptotic proteins BCL2 and BCL-XL while upregulating cleaved caspase-3. DDR restored BCL2/BCL-XL and reduced cleaved caspase-3 in a dose-dependent manner, with high-dose DDR normalizing expression to near-control levels. DDR alone was indistinguishable from controls. 4 Discussion The present study demonstrates that DDR supplementation exerts profound renoprotective effects against DOX-induced nephrotoxicity through a multifaceted mechanism involving suppression of oxidative stress, inhibition of inflammatory cascades, modulation of inflammatory signaling, restoration of antioxidant defenses, and prevention of fibrosis and apoptosis. DOX is widely used as a chemotherapeutic agent; however, its therapeutic potential is limited by severe systemic toxicities, particularly nephrotoxicity (Savani et al., 2021 ; Wu et al., 2023 ). Consistent with previous reports, we observed marked elevations in classical renal injury markers, including serum creatinine, urea, KIM-1, and NGAL, in DOX-treated mice, reflecting significant functional impairment and tubular injury. Histological assessment further revealed inflammatory infiltration, tubular necrosis, and interstitial hemorrhage. These findings recapitulate the well-characterized nephrotoxic profile of DOX, which is mediated primarily by oxidative stress, inflammation, and mitochondrial dysfunction. Importantly, DDR supplementation attenuated these alterations in a dose-dependent manner, with high-dose DDR restoring biochemical and histological parameters close to baseline. The mechanistic underpinning of DDR’s renoprotective action appears to be linked to its ability to counteract oxidative stress. DOX-induced nephrotoxicity was associated with a substantial rise in lipid peroxidation markers such as MDA, NOX-4, and PGE₂, coupled with depletion of endogenous antioxidant defenses including SOD, catalase, and GSH (Abd-Ellatif et al., 2022; Chang et al., 2023; Wu et al., 2021 ). Flavonoids pretreatment shown renal protective benefits by reducing serum levels of urea and creatinine, accompanied by enhanced renal imaging outcomes (Alsawaf et al., 2022 ; Cao et al., 2022 ). Flavonoids may have induced these effects through their antioxidant and anti-inflammatory properties, which have been previously investigated in models of chemotherapy toxicity(Farhan et al., 2023 ; Sahlan et al., 2021 ). Furthermore, flavonoids safeguarded macrophages against DOX-induced inflammation by mitigating oxidative stress and ROS (Hafez et al., 2022 ). In the present study we also found DDR supplementation effectively reversed these imbalances, markedly suppressing oxidative stress while restoring antioxidant enzyme activities. These findings indicate that DDR reinstates redox homeostasis, thereby preventing oxidative damage to renal parenchyma. The involvement of NRF2, a master regulator of cellular antioxidant responses, provides further mechanistic insight(G. Bardallo et al., 2022 ). DOX strongly suppressed NRF2 and its downstream targets HO-1 and NQO-1, whereas DDR supplementation restored their expression. This suggests that DDR activates the NRF2 pathway to augment cytoprotective antioxidant responses, an effect that is pivotal in mitigating oxidative damage. Inflammatory signaling constitutes another key axis in DOX nephrotoxicity (Wu et al., 2021 ). We have also shown a significant increase in ROS production and inflammatory mediators in renal tissues impacted by DOX, as a subsequent effect of NOX-4 activation. Excessive generation of reactive oxygen species induces oxidative damage to intracellular macromolecules and enhances the release of pro-inflammatory cytokines, ultimately worsening renal injury. The oxidative-inflammatory interaction facilitates the activation of many signaling pathways linked to cellular proliferation, apoptosis, and fibrotic remodeling (Brosius and He, 2015 ; Zhao et al., 2020). In this investigation, DOX treatment significantly elevated circulating pro-inflammatory cytokines (IL-6, TNF-α) and augmented kidney TNF-α production, indicating a severe inflammatory response. DDR treatment markedly reduced these increases, aligning with its strong antioxidant and anti-inflammatory properties. Western blot analysis further validated that DDR decreased the expression of NOX-4 and reinstated redox equilibrium, therefore mitigating oxidative stress-induced inflammation and fibrosis. These findings collectively endorse the idea that DDR alleviates DOX-induced renal damage chiefly by inhibiting ROS production and inflammatory signaling, hence safeguarding renal structure and function. Doxorubicin is well recognized for its nephrotoxic potential, with renal fibrosis being a hallmark pathological outcome. Sustained oxidative stress and inflammatory activation induced by doxorubicin drive fibroblast proliferation, extracellular matrix accumulation, and structural disruption of renal tissue, leading to progressive interstitial fibrosis and chronic kidney dysfunction (Hazem et al., 2022 ; Hu et al., 2022 ). In the present study, DDR supplementation markedly attenuated these fibrotic changes in a dose-dependent manner, with high-dose DDR restoring renal architecture close to normal. These findings highlight DDR’s potent antifibrotic activity, achieved through the suppression of oxidative and inflammatory pathways, underscoring its therapeutic promise in mitigating doxorubicin-induced renal injury. Given the central role of oxidative stress in promoting fibrogenesis, the observed antifibrotic effects of DDR are likely attributable to its suppression of this pathway, in concert with attenuation of oxidative stress and inflammation. Apoptotic signaling was also profoundly altered by DOX. We observed significant downregulation of anti-apoptotic proteins BCL2 and BCL-XL, accompanied by upregulation of cleaved caspase-3, reflecting activation of intrinsic apoptotic pathways. DDR supplementation effectively reversed these alterations, restoring BCL2 and BCL-XL expression while reducing cleaved caspase-3 levels toward baseline. This demonstrates that DDR protects renal cells from apoptosis by modulating mitochondrial apoptotic regulators. These anti-apoptotic effects complement DDR’s antioxidative and anti-inflammatory properties, together contributing to preservation of renal cellular integrity. Collectively, our findings establish that DDR confers robust renoprotection against DOX-induced nephrotoxicity through a pleiotropic mechanism. DDR attenuates oxidative stress by activating NRF2 signaling and restoring antioxidant defenses, suppresses inflammation by inhibiting ROS and cytokine release, prevents fibrotic remodeling by limiting collagen deposition, and protects against apoptosis by modulating BCL2 family proteins and caspase-3. The integrated outcome of these effects is preservation of renal architecture and function, as evidenced by normalization of biochemical markers, histological improvement, and reduced tissue injury. The translational implications of these findings are considerable. Given the clinical relevance of DOX-induced nephrotoxicity, the identification of DDR as a potential adjuvant agent offers promise for improving the safety of chemotherapy regimens. By targeting oxidative stress, inflammation, fibrosis, and apoptosis simultaneously, DDR addresses the multifactorial nature of DOX nephrotoxicity. Future studies are warranted to evaluate DDR’s pharmacokinetics, optimal dosing, and potential synergistic effects with chemotherapeutic agents in clinical settings. Furthermore, extending these investigations to chronic models of kidney injury will help determine whether DDR offers broad-spectrum renoprotection beyond chemotherapy-induced damage. 5 Conclusion In conclusion, our study provides compelling evidence that DDR supplementation exerts potent, dose-dependent renoprotective effects against DOX-induced nephrotoxicity. The mechanistic basis involves activation of NRF2-mediated antioxidant defenses, suppression of pro-inflammatory cytokines, prevention of fibrosis, and attenuation of apoptosis. These findings position DDR as a promising therapeutic candidate for mitigating renal injury in patients undergoing DOX-based chemotherapy. Declarations Conflicts of interest: The authors declare that there are no conflicts of interest. Compliance with ethics requirements This study was conducted according to the guidelines of King Faisal University and the “Executive Regulations for Research Ethics on Living Creatures (Second Edition)”, published by the National Bioethics Committee, Saudi Arabia. All animal care and experimental procedures were approved by the Animal Research Ethics Committee at King Faisal University Declaration of King Faisal University and approved by the Institutional Review Board (ETHICS3529). Funding: The authors extend their appreciation to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia, for funding this research work (Grant number: KFU252544). Author Contribution Conceptualization, P.R; methodology, validation, P.R, R.S, B.R; formal analysis, AAZ.; investigation, writing—original draft preparation, P.R.; writing—review and editing, R.S, AAZ.; visualization, B.R and G.M.B; project administration, P.R; funding acquisition, PR. All authors have read and agreed to the published version of the manuscript. Acknowledgement The authors extend their appreciation to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia. Data availability: Data is available on request from the authors. 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Cite Share Download PDF Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Molecular and Cellular Biochemistry → Version 1 posted Editorial decision: Revision requested 13 Jan, 2026 Reviews received at journal 11 Jan, 2026 Reviews received at journal 03 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers agreed at journal 28 Dec, 2025 Reviewers invited by journal 26 Dec, 2025 Editor assigned by journal 22 Dec, 2025 Submission checks completed at journal 04 Nov, 2025 First submitted to journal 04 Nov, 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. 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Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. (A) Chemical structure of 5, 4’-Dihydroxy 6,8-dimethoxy 7-O-rhamnosyl. (B) Animal experiment design. (C) Serum KIM-1 (ng/ml). (D) Serum creatinine (mg/dl). (E) Serum Urea (mg/dL). (E) Serum NGAL (ng/dL). Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, significance p \u0026lt; 0.05. * compared group I, # Compared group II, @ compared to group II.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/ee738e95f62d4fe34ef78f17.jpg"},{"id":99134498,"identity":"73e6f608-0c67-4f10-8e3d-5b2f16585703","added_by":"auto","created_at":"2025-12-29 06:18:01","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":491114,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DDR on pro-inflammatory cytokines. Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. (A) Serum level of IL-6 (pg/ml). (B) Serum level of TNF-α. (C) Activation of IL-6 and TNF-α renal tissue by western blot. Relative protein band intensities were quantified using commercial analytical software and expressed as fold change normalized to the control group. Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, significance p \u0026lt; 0.05. * compared group I, # Compared group II, @ compared to group II.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/d2bc40f315c21e9145be3662.jpg"},{"id":99315573,"identity":"f1661c1c-96df-4912-a2d2-2f64440c7f69","added_by":"auto","created_at":"2025-12-31 16:27:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":492810,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry analysis of TNF-α in renal tissue. Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. Magnification ×400 and scale bar 50µm.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/bf94c40e396c92e555831538.jpg"},{"id":99134505,"identity":"26dfbc28-2911-4d78-a6d4-ad15c5193398","added_by":"auto","created_at":"2025-12-29 06:18:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186230,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DDR on oxidative stress markers. Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. (A) MDA (nmol mg/protein), (B) Catalase (U mg/protein), (C) GSH (U mg/protein), (D) SOD (U mg/protein) (E) PGE-2 (pg/mg protein) and (F) NOX-4 (ng) (mg/protein). Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, significance p \u0026lt; 0.05. * compared group I, # Compared group II, @ compared to group II.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/d508258de88d73a392085edd.jpg"},{"id":99314943,"identity":"5b16185a-92f3-4696-b1b0-ed7419c036cf","added_by":"auto","created_at":"2025-12-31 16:24:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":346925,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DDR on NRF2, Ho-1 and NQO1 activation. . Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. (A) Activation NRF2, HO-1 and NQO1 analyzed by western blot. (B) Relative protein band intensities were quantified using commercial analytical software and expressed as fold change normalized to the control group. Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, significance p \u0026lt; 0.05. * compared group I, # Compared group II, @ compared to group II.\u003c/p\u003e","description":"","filename":"FIGURE5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/f9e27668b4dd62106bd386ee.jpg"},{"id":99134501,"identity":"403a0dd3-a2ab-43e2-8f7f-21305e8fe22c","added_by":"auto","created_at":"2025-12-29 06:18:01","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2282467,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of DDR on renal morphology. Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. Tubular region(IF), Inside blood vessel (BV red arrow),Haemorrhage in renal interstitial (black arrows) inside the renal tubules (*), vacuoles in tubular cells (blue arrows), necrotic and degenerated renal tubular cells (dRT). Wide spacing of tubules with atrophy of their lining epithelium, (green arrow). Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, significance p \u0026lt; 0.05. * compared group I, # Compared group II, @ compared to group II.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/a2a05275a7095277384dc423.jpg"},{"id":99315147,"identity":"b3568ede-4b1f-45b3-92d0-97a2a4194b5a","added_by":"auto","created_at":"2025-12-31 16:26:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":822644,"visible":true,"origin":"","legend":"\u003cp\u003eDDR inhibited renal tissue fibrosis. Group I: control, Group II: DOX alone, Group III: DOX + DDR low dose (25 mg/kg) Group IV: DOX + DDR high dose (50 mg/kg) Group V: DDR alone. Magnification ×400 and scale bar 50µm.\u003c/p\u003e","description":"","filename":"figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/a56bbbe1c441ea1b4a526504.jpg"},{"id":99134507,"identity":"2a60ece5-5623-4bcd-b163-6ab9eda69259","added_by":"auto","created_at":"2025-12-29 06:18:02","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":158262,"visible":true,"origin":"","legend":"\u003cp\u003eDDR on apoptotic markers. (A) Bcl2, Bcxl and cleaved caspase-3 analyzed by western blot. (B) Relative Protein band intensities were quantified using commercial analytical software and expressed as fold change normalized to the control group. Data are expressed as mean ± SD (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, significance p \u0026lt; 0.05. * compared group I, # Compared group II, @ compared to group II.\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/492f60894f4cf8d284a63c7d.jpg"},{"id":103251083,"identity":"38d9e31c-8633-4a70-aab7-baab2a5fc622","added_by":"auto","created_at":"2026-02-23 16:03:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6693370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8027411/v1/e144f1c2-13ae-4811-b52d-4db7e4a22ec3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Amelioration of Doxorubicin-Mediated Nephrotoxicity through Antioxidant and Anti-apoptotic Mechanisms of 5,4′-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eDoxorubicin (DOX) is a widely used anthracycline chemotherapy drug, effective against a range of cancers including breast cancer, lymphomas, and sarcomas (Almajidi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sandal et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite its clinical success, the use of DOX is often curtailed by its cumulative toxicity across various organs. While cardiac toxicity is well-documented, nephrotoxicity has increasingly been recognized as a critical limitation, particularly with extended treatment regimens (Al-Ali et al., 2024; Esp\u0026iacute;rito Santo et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to their rich blood supply and high metabolic activity, the kidneys are especially vulnerable to damage induced by DOX, primarily related to oxidative stress and inflammation.\u003c/p\u003e \u003cp\u003eThe mechanism of DOX-induced kidney injury involves overproduction of reactive oxygen species (ROS), largely mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, especially NOX-4 (Zhang et al., 2024a; Zheng et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This oxidative imbalance triggers lipid and protein damage and disrupts mitochondrial function. Concurrently, DOX promotes the release of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which drive renal inflammation (Entezari Heravi et al., 2018). Activation of the inflammatory and apoptotic responses, while suppression of the antioxidant defense pathway governed by nuclear factor erythroid 2\u0026ndash;related factor 2 (NRF2) weakens cellular resilience (Maashi et al., 2022). Collectively, these pathogenic processes result in damage to glomerular and tubular structures, interstitial fibrosis, and progressive loss of renal function.\u003c/p\u003e \u003cp\u003eNaturally occurring bioactive molecules, particularly flavonoids, have attracted considerable attention for their diverse protective effects against drug-induced organ toxicity (Rahmani et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These compounds possess strong antioxidant, anti-inflammatory, and anti-apoptotic properties, making them promising agents for kidney protection (Al-Khayri et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chagas et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Among them, 5,4\u0026prime;-dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone (DDR), a methoxylated flavonoid glycoside, has shown noteworthy pharmacological actions (Al-Saeedi and Rajendran, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; AlZahrani et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). DDR\u0026rsquo;s ability to regulate oxidative balance, modulate inflammatory mediators, and influence apoptotic pathways supports its potential in mitigating DOX-associated renal injury.\u003c/p\u003e \u003cp\u003eThis study aimed to explore the protective effects of DDR in a mouse model of chronic DOX-induced nephrotoxicity, focusing on its impact on oxidative stress, inflammation, apoptosis, and key signaling pathways including NOX-4, and NRF2. To our knowledge, this investigation provides a comprehensive analysis of the molecular mechanisms underlying DDR\u0026rsquo;s renoprotective effects against DOX toxicity.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDoxorubicin hydrochloride (analytical grade) was purchased from Sigma-Aldrich (USA). Serum urea and creatinine assay kits were obtained from BioMed and Spectrum Diagnostics (Cairo, Egypt). ELISA kits for kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were sourced from Elabscience and CUSABIO (USA). Assay kits for malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were procured from Cayman Chemical (USA). ELISA kits for tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), along with primary antibodies against TNF-α, IL-6, nuclear factor erythroid 2-related factor 2 (NRF2), B-cell lymphoma 2 (BCL2), and Caspase-3 for Western blotting, were supplied by Thermo Fisher Scientific (USA). Chromogenic substrates for immunohistochemistry (DAB), hematoxylin and eosin (H\u0026amp;E), and Masson\u0026rsquo;s trichrome staining reagents were purchased from standard histology suppliers. All other reagents used were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation of DDR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e5,4\u0026prime;-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone (DDR) was isolated from the aerial parts of \u003cem\u003eIndigofera aspalathoides\u003c/em\u003e Vahl. Plant authentication and extraction procedures followed previously established protocols (Al-Saeedi and Rajendran, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; AlZahrani et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, dried powdered plant material was subjected to methanol extraction, followed by sequential solvent fractionation. The flavonoid-rich fraction was purified via silica gel column chromatography and preparative high-performance liquid chromatography (HPLC), yielding DDR with a purity exceeding 98%, confirmed by HPLC and nuclear magnetic resonance (NMR) spectroscopy. The purified compound was stored at \u0026minus;\u0026thinsp;20\u0026deg;C in amber vials until use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental Animals\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMale Swiss albino mice (6\u0026ndash;7 weeks old, weighing 23\u0026thinsp;\u0026plusmn;\u0026thinsp;3 g) were obtained from the Experimental Surgery \u0026amp; Animal Laboratory, King Saud University, Riyadh, Saudi Arabia. Animals were acclimated for one week prior to experimentation under standard conditions: temperature 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity 60\u0026ndash;70%, and a 12-hour light/dark cycle. Animal care and use complied with the Guide for the Care and Use of Laboratory Animals (NIH) and the ARRIVE guidelines. They were housed in polypropylene cages with ad libitum access to commercial pellet diet and water. All procedures complied with institutional ethical guidelines approved by the Research Ethics Committee, King Faisal University, Hofuf, Saudi Arabia (Approval No. ETHICS3529).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental Design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFollowing acclimatization, mice were randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;6/group):\u003c/p\u003e \u003cp\u003eGroup I (Control): Received normal saline and vehicle (\u0026le;\u0026thinsp;5% DMSO in 0.5% CMC-Na) intraperitoneally for six weeks.\u003c/p\u003e \u003cp\u003eGroup II (DOX): Received DOX 2.5 mg/kg intraperitoneally, once weekly for six weeks.\u003c/p\u003e \u003cp\u003eGroup III (DOX\u0026thinsp;+\u0026thinsp;DDR Low Dose): Received DOX as above plus DDR 25 mg/kg intraperitoneally, twice weekly for six weeks.\u003c/p\u003e \u003cp\u003eGroup IV (DOX\u0026thinsp;+\u0026thinsp;DDR High Dose): Received DOX as above plus DDR 50 mg/kg intraperitoneally, twice weekly for six weeks.\u003c/p\u003e \u003cp\u003eGroup V (DDR Alone): Received DDR 50 mg/kg intraperitoneally, twice weekly for six weeks.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sample Collection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAt the end of the treatment period, mice were anesthetized with isoflurane 4%. Blood was collected by cervical decapitation, and serum was separated by centrifugation at 4000 rpm for 15 minutes. Serum samples were used for renal function tests. Kidneys were excised, rinsed with saline, and blotted dry. One kidney from each mouse was fixed in 10% neutral buffered formalin for histological and immunohistochemical analysis, while the contralateral kidney was snap-frozen at \u0026minus;\u0026thinsp;80\u0026deg;C for biochemical and Western blot assays.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Renal Function Assessment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSerum creatinine and urea levels were measured using enzymatic colorimetric kits following the manufacturers\u0026rsquo; protocols. Absorbance was recorded using a microplate reader, and concentrations were determined against calibration curves.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Measurement of Inflammatory and Renal Injury Biomarkers\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSerum levels of IL-6, TNF-α, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) were quantified using specific ELISA kits. Standards and samples were assayed in duplicates, measuring absorbance at 450 nm with 570 nm reference wavelength.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of Renal Oxidative Stress Markers\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eKidney tissues were homogenized in ice-cold 10 mM potassium phosphate buffer (pH 7.4) at a 5:1 buffer-to-tissue ratio and centrifuged at 4\u0026deg;C for 10 minutes. The supernatant was used to measure malondialdehyde (MDA) concentration via thiobarbituric acid reactive substances (TBARS) assay, and activities of reduced glutathione (GSH) and superoxide dismutase (SOD) were determined by established biochemical methods.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Quantification of NOX-4 and Prostaglandin E2 (PGE-2)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRenal homogenates were assessed for NADPH oxidase 4 (NOX-4) and prostaglandin E2 (PGE-2) concentrations using mouse-specific ELISA kits, conducted per the manufacturers\u0026rsquo; instructions.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western Blot Analysis\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e Kidney tissues were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors, followed by centrifugation at 12,000 \u0026times; g for 15 minutes at 4\u0026deg;C. Protein concentrations were determined using BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked and incubated overnight at 4\u0026deg;C with primary antibodies targeting pNFκB, NFκB, TNF-α, IL-6, IL-1β, NRF2, BCL2, and Caspase-3. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies, and protein bands were visualized using enhanced chemiluminescence. Densitometric analysis was performed with ImageJ software, normalizing protein levels to β-actin controls.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Histopathological Examination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFormalin-fixed kidneys were processed, paraffin-embedded, sectioned at 5 \u0026micro;m thickness, and stained with hematoxylin and eosin (H\u0026amp;E) for general morphology.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Immunohistochemistry\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRenal sections were deparaffinized, rehydrated, and endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. Non-specific binding was blocked with normal goat serum. Sections were incubated overnight at 4\u0026deg;C with anti-TNF-α antibody (1:100 dilution), followed by incubation with HRP-conjugated secondary antibody. The antigen-antibody complexes were visualized using DAB substrate.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Masson\u0026rsquo;s Trichrome Staining\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eKidney sections (5 \u0026micro;m) were stained with Masson\u0026rsquo;s trichrome to assess collagen deposition and fibrosis. The staining protocol included sequential incubation with Weigert\u0026rsquo;s iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue. Stained sections were dehydrated, cleared, and mounted. Collagen content was quantified by calculating the percentage of blue-stained area using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test using GraphPad Prism software. Differences were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of DDR on kidney function\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe chemical structure of DDR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and experimental design (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) are shown. DOX administration significantly elevated serum KIM-1, creatinine, urea, and NGAL compared with controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;F). DOX markedly elevated serum KIM-1 compared with controls, while DDR reduced levels dose-dependently, with the high dose restoring near baseline. Serum creatinine increased robustly in DOX mice, which DDR significantly attenuated, particularly at high dose. Urea followed a similar pattern, with DDR lowering levels close to controls. NGAL was also dramatically increased in DOX mice, but DDR normalized values in a dose-dependent manner. DDR alone did not differ from controls. DDR supplementation reduced all markers in a dose-dependent manner, with high-dose DDR restoring values close to baseline. DDR alone had no adverse effect, confirming its safety.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of DDR on inflammatory cytokines\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDOX markedly increased circulating IL-6 and TNF-α (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). IL-6 levels varied significantly among groups. DOX induced a robust increase, while DDR significantly reduced IL-6 in both low- and high-dose groups, with the latter restoring levels to near baseline. A similar trend was observed for TNF-α, with high-dose DDR providing the greatest suppression. DDR significantly suppressed these elevations, with the high dose normalizing cytokine levels. DDR alone was indistinguishable from controls.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Renal Expression of TNF-α and IL-6\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWestern blot (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D) and immunohistochemistry TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) confirmed strong upregulation of TNF-α and IL-6 in DOX-treated kidneys. DDR reduced both proteins dose-dependently, with high-dose treatment restoring near-control levels. Western blot analysis confirmed marked upregulation of TNF-α in DOX-treated kidneys. DDR supplementation significantly and dose-dependently reduced expression, with high-dose treatment restoring values close to baseline. Immunohistochemistry supported these findings: DOX kidneys exhibited group showing positive immunostaining of the glomerulus (red arrow) and tubules (block arrow), whereas DDR reduced staining in a dose-dependent manner. DDR alone showed no difference from controls.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of DDR on oxidative stress\u003c/h2\u003e \u003cp\u003eDOX induced severe oxidative stress, reflected by increased MDA, PGE₂, and NOX-4, and depletion of catalase, GSH, and SOD (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;F). DOX significantly increased renal MDA, and NOX-4 while reducing catalase, GSH, and SOD. DDR supplementation significantly reversed these alterations in a dose-dependent fashion, with high-dose DDR restoring all parameters near baseline. DDR alone remained comparable to controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of DDR on NRF2/HO-1/NQO-1 Pathway\u003c/h2\u003e \u003cp\u003eDOX markedly suppressed NRF2, HO-1, and NQO-1 expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). DOX profoundly suppressed NRF2, and NQO-1. DDR treatment restored expression in a dose-dependent manner, with high-dose DDR normalizing all three proteins. DDR alone slightly elevated HO-1 above baseline but otherwise showed no deviation from controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Histopathological Alterations\u003c/h2\u003e \u003cp\u003eControl kidneys displayed intact glomeruli and tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). DOX caused severe pathology, including lymphocytic infiltration, hemorrhage, vacuolization, necrosis, tubular dilation, and epithelial atrophy. DDR afforded dose-dependent protection: low-dose DDR partially ameliorated injury, while high-dose DDR preserved architecture with minimal inflammation and necrosis. DDR alone maintained normal histology, verifying its safety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Effect of DDR on renal fibrosis\u003c/h2\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining revealed extensive interstitial fibrosis, perivascular thickening, and collagen deposition in DOX kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). DDR supplementation reduced collagen deposition in a dose-dependent manner, with high-dose DDR showing marked antifibrotic protection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Apoptotic Markers\u003c/h2\u003e \u003cp\u003eDOX decreased anti-apoptotic BCL2 and BCL-XL while elevating cleaved caspase-3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, B). DOX suppressed anti-apoptotic proteins BCL2 and BCL-XL while upregulating cleaved caspase-3. DDR restored BCL2/BCL-XL and reduced cleaved caspase-3 in a dose-dependent manner, with high-dose DDR normalizing expression to near-control levels. DDR alone was indistinguishable from controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe present study demonstrates that DDR supplementation exerts profound renoprotective effects against DOX-induced nephrotoxicity through a multifaceted mechanism involving suppression of oxidative stress, inhibition of inflammatory cascades, modulation of inflammatory signaling, restoration of antioxidant defenses, and prevention of fibrosis and apoptosis. DOX is widely used as a chemotherapeutic agent; however, its therapeutic potential is limited by severe systemic toxicities, particularly nephrotoxicity (Savani et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Consistent with previous reports, we observed marked elevations in classical renal injury markers, including serum creatinine, urea, KIM-1, and NGAL, in DOX-treated mice, reflecting significant functional impairment and tubular injury. Histological assessment further revealed inflammatory infiltration, tubular necrosis, and interstitial hemorrhage. These findings recapitulate the well-characterized nephrotoxic profile of DOX, which is mediated primarily by oxidative stress, inflammation, and mitochondrial dysfunction. Importantly, DDR supplementation attenuated these alterations in a dose-dependent manner, with high-dose DDR restoring biochemical and histological parameters close to baseline.\u003c/p\u003e \u003cp\u003eThe mechanistic underpinning of DDR\u0026rsquo;s renoprotective action appears to be linked to its ability to counteract oxidative stress. DOX-induced nephrotoxicity was associated with a substantial rise in lipid peroxidation markers such as MDA, NOX-4, and PGE₂, coupled with depletion of endogenous antioxidant defenses including SOD, catalase, and GSH (Abd-Ellatif et al., 2022; Chang et al., 2023; Wu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Flavonoids pretreatment shown renal protective benefits by reducing serum levels of urea and creatinine, accompanied by enhanced renal imaging outcomes (Alsawaf et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Flavonoids may have induced these effects through their antioxidant and anti-inflammatory properties, which have been previously investigated in models of chemotherapy toxicity(Farhan et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sahlan et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, flavonoids safeguarded macrophages against DOX-induced inflammation by mitigating oxidative stress and ROS (Hafez et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study we also found DDR supplementation effectively reversed these imbalances, markedly suppressing oxidative stress while restoring antioxidant enzyme activities. These findings indicate that DDR reinstates redox homeostasis, thereby preventing oxidative damage to renal parenchyma. The involvement of NRF2, a master regulator of cellular antioxidant responses, provides further mechanistic insight(G. Bardallo et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). DOX strongly suppressed NRF2 and its downstream targets HO-1 and NQO-1, whereas DDR supplementation restored their expression. This suggests that DDR activates the NRF2 pathway to augment cytoprotective antioxidant responses, an effect that is pivotal in mitigating oxidative damage. Inflammatory signaling constitutes another key axis in DOX nephrotoxicity (Wu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We have also shown a significant increase in ROS production and inflammatory mediators in renal tissues impacted by DOX, as a subsequent effect of NOX-4 activation. Excessive generation of reactive oxygen species induces oxidative damage to intracellular macromolecules and enhances the release of pro-inflammatory cytokines, ultimately worsening renal injury. The oxidative-inflammatory interaction facilitates the activation of many signaling pathways linked to cellular proliferation, apoptosis, and fibrotic remodeling (Brosius and He, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhao et al., 2020). In this investigation, DOX treatment significantly elevated circulating pro-inflammatory cytokines (IL-6, TNF-α) and augmented kidney TNF-α production, indicating a severe inflammatory response. DDR treatment markedly reduced these increases, aligning with its strong antioxidant and anti-inflammatory properties. Western blot analysis further validated that DDR decreased the expression of NOX-4 and reinstated redox equilibrium, therefore mitigating oxidative stress-induced inflammation and fibrosis. These findings collectively endorse the idea that DDR alleviates DOX-induced renal damage chiefly by inhibiting ROS production and inflammatory signaling, hence safeguarding renal structure and function.\u003c/p\u003e \u003cp\u003eDoxorubicin is well recognized for its nephrotoxic potential, with renal fibrosis being a hallmark pathological outcome. Sustained oxidative stress and inflammatory activation induced by doxorubicin drive fibroblast proliferation, extracellular matrix accumulation, and structural disruption of renal tissue, leading to progressive interstitial fibrosis and chronic kidney dysfunction (Hazem et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, DDR supplementation markedly attenuated these fibrotic changes in a dose-dependent manner, with high-dose DDR restoring renal architecture close to normal. These findings highlight DDR\u0026rsquo;s potent antifibrotic activity, achieved through the suppression of oxidative and inflammatory pathways, underscoring its therapeutic promise in mitigating doxorubicin-induced renal injury. Given the central role of oxidative stress in promoting fibrogenesis, the observed antifibrotic effects of DDR are likely attributable to its suppression of this pathway, in concert with attenuation of oxidative stress and inflammation. Apoptotic signaling was also profoundly altered by DOX. We observed significant downregulation of anti-apoptotic proteins BCL2 and BCL-XL, accompanied by upregulation of cleaved caspase-3, reflecting activation of intrinsic apoptotic pathways. DDR supplementation effectively reversed these alterations, restoring BCL2 and BCL-XL expression while reducing cleaved caspase-3 levels toward baseline. This demonstrates that DDR protects renal cells from apoptosis by modulating mitochondrial apoptotic regulators. These anti-apoptotic effects complement DDR\u0026rsquo;s antioxidative and anti-inflammatory properties, together contributing to preservation of renal cellular integrity.\u003c/p\u003e \u003cp\u003eCollectively, our findings establish that DDR confers robust renoprotection against DOX-induced nephrotoxicity through a pleiotropic mechanism. DDR attenuates oxidative stress by activating NRF2 signaling and restoring antioxidant defenses, suppresses inflammation by inhibiting ROS and cytokine release, prevents fibrotic remodeling by limiting collagen deposition, and protects against apoptosis by modulating BCL2 family proteins and caspase-3. The integrated outcome of these effects is preservation of renal architecture and function, as evidenced by normalization of biochemical markers, histological improvement, and reduced tissue injury. The translational implications of these findings are considerable. Given the clinical relevance of DOX-induced nephrotoxicity, the identification of DDR as a potential adjuvant agent offers promise for improving the safety of chemotherapy regimens. By targeting oxidative stress, inflammation, fibrosis, and apoptosis simultaneously, DDR addresses the multifactorial nature of DOX nephrotoxicity. Future studies are warranted to evaluate DDR\u0026rsquo;s pharmacokinetics, optimal dosing, and potential synergistic effects with chemotherapeutic agents in clinical settings. Furthermore, extending these investigations to chronic models of kidney injury will help determine whether DDR offers broad-spectrum renoprotection beyond chemotherapy-induced damage.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn conclusion, our study provides compelling evidence that DDR supplementation exerts potent, dose-dependent renoprotective effects against DOX-induced nephrotoxicity. The mechanistic basis involves activation of NRF2-mediated antioxidant defenses, suppression of pro-inflammatory cytokines, prevention of fibrosis, and attenuation of apoptosis. These findings position DDR as a promising therapeutic candidate for mitigating renal injury in patients undergoing DOX-based chemotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest:\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompliance with ethics requirements\u003c/strong\u003e \u003cp\u003eThis study was conducted according to the guidelines of King Faisal University and the \u0026ldquo;Executive Regulations for Research Ethics on Living Creatures (Second Edition)\u0026rdquo;, published by the National Bioethics Committee, Saudi Arabia. All animal care and experimental procedures were approved by the Animal Research Ethics Committee at King Faisal University Declaration of King Faisal University and approved by the Institutional Review Board (ETHICS3529).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe authors extend their appreciation to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia, for funding this research work (Grant number: KFU252544).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, P.R; methodology, validation, P.R, R.S, B.R; formal analysis, AAZ.; investigation, writing\u0026mdash;original draft preparation, P.R.; writing\u0026mdash;review and editing, R.S, AAZ.; visualization, B.R and G.M.B; project administration, P.R; funding acquisition, PR. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors extend their appreciation to the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eData is available on request from the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Khayri JM, Sahana GR, Nagella P, Joseph BV, Alessa FM, Al-Mssallem MQ (2022) Flavonoids as Potential Anti-Inflammatory Molecules: A Review. Molecules (Basel Switzerland) 27\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Saeedi F, Rajendran P (2024) Anti-metastasis activity of 5,4'-dihydroxy 6,8-dimethoxy 7-O-rhamnosyl flavone from Indigofera aspalathoides Vahl on breast cancer cells. 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Legends\u003c/span\u003e\u003c/li\u003e\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":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Doxorubicin, NOX-4, NFκB, nephrotoxicity, flavonoids","lastPublishedDoi":"10.21203/rs.3.rs-8027411/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8027411/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDoxorubicin (DOX) is a potent chemotherapeutic agent whose clinical utility is limited by cumulative nephrotoxicity driven by oxidative stress, inflammation, and apoptosis. Natural flavonoids have shown promise in mitigating such adverse effects. This study evaluated the renoprotective efficacy of 5,4\u0026prime;-dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone (DDR) against chronic DOX-induced kidney injury, focusing on the involvement of NOX-4-mediated oxidative stress, inflammatory signalling, NRF2 antioxidant pathways, and apoptotic regulators BCL2 and Caspase-3.Male albino mice were randomized into five groups (n\u0026thinsp;=\u0026thinsp;6): control, DOX (2.5 mg/kg intraperitoneally, once weekly for six weeks), DOX plus low-dose DDR (25 mg/kg, twice weekly), DOX plus high-dose DDR (50 mg/kg, twice weekly), and DDR alone. Renal function was assessed via serum creatinine, urea, and biomarkers NGAL and KIM-1. Oxidative stress markers (MDA, GSH, SOD, CAT), pro-inflammatory cytokines (pNFκB,TNF-α, IL-6 and IL-1β), and protein expression of NOX-4, NRF2, BCL2, and Caspase-3 were quantified by biochemical assays, ELISA, immunohistochemistry, and Western blotting. Kidney tissues underwent histopathological evaluation using hematoxylin and eosin and Masson\u0026rsquo;s trichrome staining.\u003c/p\u003e \u003cp\u003eDOX administration induced significant renal impairment, characterized by elevated MDA, NOX-4, pNFκB,TNF-α, IL-6, Il-1β and Caspase-3 levels, alongside reduced antioxidant enzyme activities and BCL2 expression. Activation of signaling and suppression of NRF2 correlated with marked glomerular and tubular injury. DDR treatment ameliorated these effects in a dose-dependent manner; high-dose DDR significantly improved renal function, diminished oxidative and inflammatory mediators, enhanced antioxidant defenses, suppressed NOX-4 and upregulated NRF2 and BCL2, resulting in preserved renal histology.\u003c/p\u003e \u003cp\u003eDDR confers dose-dependent nephroprotection against chronic DOX-induced toxicity by modulating oxidative stress, inflammation, and apoptosis. These findings advocate DDR\u0026rsquo;s potential as an adjunctive agent to alleviate chemotherapy-associated renal damage.\u003c/p\u003e","manuscriptTitle":"Amelioration of Doxorubicin-Mediated Nephrotoxicity through Antioxidant and Anti-apoptotic Mechanisms of 5,4′-Dihydroxy-6,8-dimethoxy-7-O-rhamnosylflavone","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-29 06:17:57","doi":"10.21203/rs.3.rs-8027411/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T11:37:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T07:39:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-03T21:28:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12484565917671786234904853779588869864","date":"2025-12-29T11:44:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166469280654653158538295809633721555187","date":"2025-12-28T08:10:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-26T06:37:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-22T13:12:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-04T16:59:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular and Cellular Biochemistry","date":"2025-11-04T09:40:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-and-cellular-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcbi","sideBox":"Learn more about [Molecular and Cellular Biochemistry](https://www.springer.com/journal/11010)","snPcode":"11010","submissionUrl":"https://submission.nature.com/new-submission/11010/3","title":"Molecular and Cellular Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7f35c5f9-3760-4ccb-9280-8488349e6983","owner":[],"postedDate":"December 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:01:14+00:00","versionOfRecord":{"articleIdentity":"rs-8027411","link":"https://doi.org/10.1007/s11010-026-05506-0","journal":{"identity":"molecular-and-cellular-biochemistry","isVorOnly":false,"title":"Molecular and Cellular Biochemistry"},"publishedOn":"2026-02-21 15:57:39","publishedOnDateReadable":"February 21st, 2026"},"versionCreatedAt":"2025-12-29 06:17:57","video":"","vorDoi":"10.1007/s11010-026-05506-0","vorDoiUrl":"https://doi.org/10.1007/s11010-026-05506-0","workflowStages":[]},"version":"v1","identity":"rs-8027411","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8027411","identity":"rs-8027411","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}