The potential therapeutic effect of quercetin on mitochondrial dysfunction in hepatorenal toxicity induced by aluminum chloride in an experimental rat modelDownload PDF Download PDF ArticleOpen accessPublished: 03 August 2026Tasneem N. Hafez1,Magda A. Megahed1,Bothaina F. Mahmoud1,Mohammed Salama2 &…Nesma A. Ghazal1 Scientific Reports volume 16, Article number: 23767 (2026) Cite this articleSave articleView saved researchAbstractAluminum is a xenobiotic element known to induce hepatorenal toxicity through mechanisms involving mitochondrial dysfunction, oxidative stress, and inflammation. Quercetin, a dietary flavonoid with potent antioxidant and anti-inflammatory properties, has shown promise as a therapeutic agent. This study aimed to evaluate the potential therapeutic effects of quercetin against aluminum chloride (AlCl₃)-induced hepatorenal toxicity and mitochondrial dysfunction in rats. Hepatorenal toxicity was induced by oral administration of hydrated aluminum chloride (75 mg/kg body weight) daily for six weeks. Quercetin was administered intraperitoneally at a dose of 30 mg/kg body weight daily for four weeks. Biochemical assays, mitochondrial gene expression analysis, and histopathological examinations were conducted to assess the therapeutic effects. Quercetin significantly ameliorated lipid, protein, and DNA oxidation parameters (MDA, AOPPs and 8-OHdG respectively), reduced inflammation marker (TNF-α), and restored mitochondrial biogenesis markers, including PGC-1α, mtTFA and mitochondrial DNA copy number (mtDNA-CN). In addition, Quercetin significantly decreased TNF-α and increased PGC-1α contents at protein levels. Histopathological findings corroborated these results, demonstrating that quercetin improved liver and kidney architecture. These findings suggest that quercetin may serve as a potential therapeutic agent for aluminum-induced hepatorenal toxicity.SubjectsBiochemistryDiseasesDrug discoveryMedical researchMolecular biologyPhysiologyIntroductionAluminum chloride (AlCl₃) exposure poses a significant environmental and occupational health risk due to its toxic effects on vital organs, particularly the liver and kidneys. The mechanisms underlying AlCl₃-induced toxicity include oxidative stress, mitochondrial dysfunction, and impaired antioxidant defenses1.In the liver, AlCl₃ exposure disrupts mitochondrial energy metabolism, elevates serum liver enzymes, and induces histopathological changes such as necrosis and fibrosis. Similarly, in the kidneys, AlCl₃ causes tubular necrosis, fibrosis, and epithelial hyperplasia, further exacerbating organ dysfunction2.The master regulator of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α), controls the expression of mtTFA which facilitate the transcription, replication of mitochondrial DNA (mtDNA) and mitochondrial biogenesis3.Mitochondrial dysfunction is central to the pathogenesis of hepatorenal toxicity, as mitochondria are critical for energy production and cellular homeostasi4. Disruption of mitochondrial biogenesis contributes to decreased ATP production and increased reactive oxygen species (ROS), leading to cellular damage5. Following oxidative stress exposure, Nrf2 is phosphorylated and Keap1 becomes inactive. When phosphorylateyod Nrf2 (p-Nrf2) accumulates in the nucleus, it binds with the antioxidant-response element (ARE) and activates a variety of genes, including those that produce transport molecules, detoxifying enzymes, and antioxidants6. Extensive evidence highlights a reciprocal regulatory feedback loop between PGC-1α and Nrf2, wherein Nrf2 directly drives mitochondrial biogenesis and cross-talks with PGC-1α to promote lifespan extension7.Quercetin, a naturally occurring flavonoid, has garnered attention for its antioxidant and anti-inflammatory properties. It neutralizes free radicals, reduces oxidative stress, and modulates biological pathways involved in inflammation and mitochondrial function. Recent studies have highlighted its protective effects against renal inflammation, ferroptosis, and apoptosis8.Given the pivotal role of mitochondrial dysfunction in AlCl₃ induced toxicity, this study investigates the therapeutic potential of quercetin in mitigating mitochondrial damage and hepatorenal toxicity in an experimental rat model.Materials and methodsExperimental animalsA total number of 32 two months Wistar male albino rats, (100-150g) were used. The animals were obtained from the animal house of Medical Research Institute, Alexandria University, Egypt. Rats were housed in standard cages in well-ventilated rooms (25 ± 2 °C), with a relative humidity of (43 ± 3), with free access to water and food and 12 hours’ light/dark cycle before experimentation.Ethical statementAll experiments pursued the standards of the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978) and were performed after the approval of the Institutional Animal Care and Use Committee (IACUC)-Alexandria University, Egypt (Approval No.: AU01223101512). The study also followed ARRIVE guidelines and complied with the National Research Council’s Guide for the Care and Use of Laboratory Animals.Induction of hepatorenal toxicityHepatorenal toxicity was induced in rats using hydrated aluminum chloride (AlCl3.6H2O) solution that was given orally at a dose of 75 mg/kg body weight daily for 6 consecutive weeks9Treatment with quercetinQuercetin obtained from Sigma Aldrich was administrated intraperitoneally to rats as a powder dissolved in water at a dose of 30 mg/kg body weight daily for 4 weeks10.Experimental designasThe animals were given standard food and water ad-libitum. Rats were classified into four groups each group contains 8 rats: Group I (Control group), normal healthy male rats. Group II (Quercetin control group), rats were received a daily intraperitoneal injection of quercetin (30 mg/kg body weight, dissolved in 0.25% v/v DMSO) for 4 weeks10. Group III (Untreated AlCl3group), rats were administered aluminum chloride (AlCl3.6H2O) solution (75 mg/kg body weight /day)9 orally for 6 consecutive weeks followed by daily intraperitoneal injections of the DMSO vehicle alone for 4 weeks. Group IV (Quercetin-treated AlCl3group), rats were administered AlCl3.6H2O solution (75 mg/kg body weight/day) orally for 6 consecutive weeks then treated interperitoneally wih quercetin (30 mg/kg body weight, dissolved in 0.25% v/v DMSO) daily for 4 weeks.Collection of samplesAfter 24 hours from the last administration, rats in all groups were sacrificed under deep anesthesia via isofluoran inhalation. The blood samples were collected from dorsal vein into serum gel separator tubes from each rat. The samples were left f or 20 min at 4◦C, centrifuged at 3000 xg for 10 minutes using Hettich Zentrifugen Tuttlingen centrifuge to obtain serum. Sera were stored at −80◦C until used for assessment liver function tests (ALT, AST, ALP, total bilirubin), kidney function tests (urea, creatinine), lipid profile parameters (total cholesterol, triglycerides, HDL-C, LDL-C), and advanced oxidation protein products (AOPPs).The excised liver and kidney tissues were rinsed with saline and then divided into two halves. The first half was divided into two parts. First part of excised tissue was homogenized in phosphate buffer saline (PBS) pH 7.4 in the ratio of 1:10 (0.125 gm of tissue in 1.25 ml PBS). The homogenate was used for the determination of malondialdehyde (MDA) while the second aliquot was centrifuged at 10000 rpm, at 4°C for 20 minutes and the obtained supernatants were used for the determination of 8-hydroxy guanidine (8-OHdG), Peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1 α) and tumor necrosis factor alpha (TNFα) by ELISA. Second part of excised tissue was used for the extraction of total RNA for Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis for assessment of gene expression of PGC-1 α, mitochondrial transcription factor A (mtTFA), nuclear factor-erythroid 2-related factor 2 (Nrf2) and TNF-α and extraction of DNA for determination of mtDNA-CN. Second half of liver and kidney tissues was fixed in 10 % buffered formalin for histological examination.Histopathological examinationFollowing necropsy, liver and kidney specimens were immediately fixed in phosphate-buffered formalin (10%, pH 7.4) for at least 24 hours which were then processed using conventional paraffin embedding technique11 Sections of 5 μm thick were sliced, mounted on slides deparaffinated in xylene and rehydrated using decreasing concentrations of ethanol. Slides were stained with hematoxylin and eosin (H&E) for routine histopathological setting. Stained sections were blindly evaluated using light microscope (Leica, DM500) and photographed at a magnification of ×400 using a digital camera (EC3, Leica, Germany). The histopathological staging (or scoring) was done as a semi-quantitative assessment. This scoring is based on a standard 0 to 3 scale , evaluating the percentage of tissue damage or alteration observed across multiple microscopic fields (usually graded as: 0 = Normal, 1 = Mild [50%]). For Liver Tissue Staging Parameters, the scoring for liver slides evaluates the degree of tissue injury based on two main criteria: Hepatocyte vacuolation and degeneration. For Kidney Tissue Staging Parameters, the renal scoring evaluates the deformity, shrinkage, or congestion of the glomeruli and the widening/loss of Bowman’s space.Serum parameters measurementsThe blood samples were obtained and assayed for liver function parameters according to the manufacturer’s instructions using serum ALT Bio-Med Diagnostic INC (USA) kit (ALT Cat. No.: 1200)12, serum AST Bio-Med Diagnostic INC (USA) kit (AST Cat. No.: 1202)12, serum ALP Bio-Med Diagnostic INC (USA) kit (ALP Cat. No.: 101090)13, and serum total bilirubin spectrum Diagnostic (Germany) kit (Cat. No.: 222 001)14.Also, kidney function parameters were assayed according to the manufacturer’s instructions using serum urea Bio-Med Diagnostic INC (USA) kit (Cat. No.: IFUFCC40)15, and serum creatinine Bio-Med Diagnostic INC (USA) kit (Cat. No.: IFUFCC09)16.In addition, lipid profile was investigated by using cholesterol Agappe Diagnostic LTD (India) kit17, serum triglycerides Agappe Diagnostic LTD (India) kit13 and serum HDL-C level Agappe Diagnostic LTD (India) kit18. Serum LDL–C was calculated19.Determination of AOPPs levels according to the manufacturer’s instructions using AOPPs ELISA kit (Cat. No.: CSBEQ027429RA)20.Determination of malondialdehyde (MDA) contentMalondialdehyde was determined according to the method of21. The tissue samples were heated with thiobarbituric acid (TBA) at low pH. The resulting pink chromogen has a maximal absorbance at 532 nm.Protein content determination of 8-OHdG, PGC1α, and TNF-α by ELISA.The content of rat 8-OHdG, PGC1α, and TNF-α in the samples were measured by ELISA kit (Cat. No.: CSB-E10526r)22, (Cat. No.: MBS1600735)23, and (Cat. No.: CSB-E11987r)24. respectively.Determination of total protein contentsA modification method of Lowry et al.25 was used for the determination of protein in the samples.Gene expression analysisThirty mg of kidney tissues were used for total RNA extraction using Gene Direx Kit (USA) (Cat. No.: NA021-0100) according to the manufacturer’s instructions. The concentration and integrity of extracted RNA were checked using nanodrop. The reverse transcription of the extracted RNA was performed by Viva cDNA Synthesis Kit (vivantis) according to the manufacturer instructions. The tissues expression of PGC-1α, mtTFA, Nrf2, and TNF-α were quantified in the cDNA by CFX Maestro™ Software (Bio-Rad, USA) using Rotor-Gene SYBR Green PCR Kit (Qiagen®, Germany). The housekeeping gene 18S rRNA was used as a reference gene for normalization. The primers used for the determination of rat genes are presented in Table 1. The relative change in mRNA expression in samples was calculated using the 2-ΔΔCt method26.Table 1 Primer sets of PGC-1α, mtTFA, Nrf2, TNF-α, and 18S rRNA.Full size tableMitochondrial DNA copy number determinationA qRT-PCR assay was developed to estimate relative mtDNA copy number (mtDNA-CN) by comparing PCR amplicons of mitochondrial DNA to a single nuclear gene. Following genomic DNA isolation, specific primer pairs for mtDNA and nuclear PGC-1α were used in equal PCR cycles to calculate the mtDNA signal relative to nuclear DNA. The mtDNA content is expressed as the ratio Ct (mtDNA)/Ct (nDNA), where lower Ct values indicate higher template concentration. This ratio demonstrates that increasing Ct values correlate with a decrease in mtDNA per cell, ultimately representing mtDNA-CN as log R, where R=2 –ΔCt and ΔCt= Ct mtDNA – Ct nDNA27 Table 2.Table 2 Primers for nuclear PGC-1α and mtDNA for qRT-PCR.Full size tableStatistical analysisData were analyzed using SPSS software package version 18.0 (SPSS Chicago, IL, USA). The data were expressed as means ± SD and analyzed using a one- way analysis of variance (ANOVA) and followed by post hoc Tukey test to compare the mean values between and within treated groups compared to untreated and control groups. Differences were considered statistically significant at p value < 0.05. Correlation studies were performed using Pearson’s correlation coefficient28.ResultsLiver function testsThe AlCl3 exposure significantly increased serum ALT, AST, ALP activities and total bilirubin levels compared to controls (p ≤ 0.05). Treatment with quercetin significantly reduced these parameters, though not to control levels (p ≤ 0.05). The quercetin control group exhibited lower ALP and total bilirubin levels compared to the untreated AlCl3 group, indicating quercetin’s protective role in liver function (Table 3).Table 3 Statistical analysis of serum liver function tests in the different studied groups.Full size tableKidney function testsAs shown in (Table 4), AlCl3 exposure significantly raised urea and creatinine levels (p ≤ 0.05). Quercetin treatment reduced these levels significantly compared to untreated AlCl3-exposed rats, though levels remained elevated compared to the control group (p ≤ 0.05). Quercetin alone showed no adverse effects on kidney function.Table 4 Statistical analysis of serum kidney function tests in the different studied groups.Full size tableLipid profile parametersAs seen in (Table 5), Total cholesterol, triglycerides, and LDL-C levels were significantly elevated in AlCl3-exposed rats, while HDL-C levels were significantly decreased (p ≤ 0.05). Quercetin treatment significantly reduced cholesterol, triglycerides, and LDL-C levels while improving HDL-C levels, restoring parameters closer to control values (p ≤ 0.05).Table 5 Statistical analysis of lipid profile parameters in the different studied groups.Full size tableSerum advanced oxidation protein products (AOPPs)As seen in (Table 6), The AlCl3 exposed group exhibited a significantly higher AOPPs level as compared to control groups (p ≤ 0.05). Quercetin treatment significantly reduced AOPPs though it remained higher than the control group (p ≤ 0.05).Table 6 Statistical analysis of serum AOPPs levels (nmol/ml) in the different studied groups.Full size tableHepatic and renal Malondialdehyde (MDA)As seen in (Table 7), The AlCl3 exposed group exhibited a significantly higher MDA level as compared to control groups (p ≤ 0.05). Quercetin treatment significantly reduced MDA though it remained higher than the control group (p ≤ 0.05).Table 7 Statistical analysis of MDA contents (nmol/mg protein) in the different studied groups.Full size tableHepatic and renal 8-hydroxy guanosine (8-OHdG) in ratsAs seen in (Table 8), The AlCl3 exposed group exhibited a significantly higher 8-OHdG level as compared to control groups (p ≤ 0.05). Quercetin treatment significantly reduced 8-OHdG though it remained higher than the control group (p ≤ 0.05).Table 8 Statistical analysis of hepatic and renal 8-OHdG contents (ng/mg protein) in the different studied groups.Full size tableHepatic and renal peroxisome proliferator activator receptor gamma-coactivator 1α (PGC-1α) contents in ratsAs shown in (Table 9), the AlCl3 exposed group exhibited a significantly hepatic and renal lower PGC-1α contents as compared to control and quercetin control groups (p ≤ 0.05). Quercetin control rats showed a statistically significant hepatic reduction but renal elevation in PGC-1α contents as compared to control rats (p ≤ 0.05). AlCl3 exposed group treated with quercetin showed a statistically significant hepatic elevation in PGC-1α contents as compared to untreated AlCl3 exposed rats but statistically significant decline in hepatic PGC-1α as compared to control and quercetin control groups (p ≤ 0.05). In case of renal tissue, there was statistically significant elevation in PGC-1α contents in quercetin treated AlCl3 group as compared to untreated AlCl3 group (p ≤ 0.05), but not significant difference compared to control and quercetin control groups.Table 9 Statistical analysis of hepatic and renal PGC-1α content (ng/mg protein) in the different studied groups.Full size tableHepatic and renal tumor necrosis factor alpha (TNF-α) contents in ratsAs shown in (Table 10), the AlCl3 exposed group exhibited a significantly hepatic and renal higher TNF-α contents as compared to control and quercetin control groups (p ≤ 0.05). AlCl3 exposed group treated with quercetin showed a statistically significant hepatic and renal reduction in TNF-α contents as compared to untreated AlCl3 exposed rats but statistically significant increased compared with control and quercetin control group (p ≤ 0.05).Table 10 Statistical analysis of hepatic and renal TNF-α contents (pg/mg protein) in the different studied groups.Full size tableHepatic and renal expression of PGC-1α, mtTFA, Nrf2, and TNF-αAs shown in (Figs. 1,2,3,4,5,6,7,8), AlCl3 exposure led to significant downregulation of genes related to mitochondrial function, such as PGC-1α, mtTFA, and Nrf2, while upregulating TNF-α expression (p ≤ 0.05). Quercetin treatment significantly restored the expression of these genes toward control levels (p ≤ 0.05), indicating its role in mitigating mitochondrial dysfunction and inflammation.Fig. 1Full size imageHepatic PGC-1 α content (ng/mg protein) in the different studied groups.Fig. 2Full size imageRenal PGC-1 α content (ng/mg protein) in the different studied groups.Fig. 3Full size imageHepatic mtTFA gene expression (Fold change) in the different studied groups.Fig. 4Full size imageRenal mtTFA gene expression (Fold change) in the different studied groups.Fig. 5Full size imageHepatic Nrf2 gene expression (Fold change) in the different studied groups.Fig. 6Full size imageRenal Nrf2 gene expression (Fold change) in the different studied groups.Fig. 7Full size imageHepatic TNF-α gene expression (Fold change) in the different studied groups.Fig. 8Full size imageRenal TNF-α gene expression (Fold change) in the different studied groups.As shown in (Figs. 9,10), Mitochondrial DNA copy number (mtDNA-CN) was significantly reduced in AlCl3-exposed rats (p ≤ 0.05), but treatment with quercetin significantly increased mtDNA-CN levels (p ≤ 0.05).Fig. 9Full size imageHepatic mtDNA-CN in the different studied groups.Fig. 10Full size imageRenal mtDNA-CN in the different studied groups.Correlation studiesCorrelation between different parameters in hepatic tissues in quercetin-treated AlCl3 groupTumer necrosis factor-α (TNF-α) gene expression was positively correlated with Nrf2 gene expression (r= 0.886, p