Elevated uric acid levels have been consistently linked to worsening kidney function in clinical studies (Chen et al., 2020). To better understand this relationship and evaluate potential treatments, we established the experimental design outlined in Figure 1. Our assessment of renal function included multiple clinical parameters: serum uric acid, BUN and creatinine levels, urinary albumin/creatinine ratios, and systemic blood pressure measurements. The animal model successfully replicated key features of hyperuricemia, with treated rats showing marked kidney dysfunction and hypertension compared to normal controls.

In our therapeutic evaluation, we tested quercetin at two different concentrations (50 and 100 mg/kg daily), alongside febuxostat as an established reference treatment, with doses converted based on body surface area normalization between humans and rats (Di Pierro et al., 2025; Wang et al., 2012; Long et al., 2016; Sanchez-Lozada et al., 2008). The results demonstrated quercetin’s dose-dependent renal benefits, with both concentrations showing significant improvements across all measured parameters - including serum BUN, creatinine, ACR, and blood pressure normalization (Figures 2A–D). Notably, the higher quercetin dose exerted similar therapeutic effects to febuxostat, although to a lesser extent.

Notably, quercetin administration showed an excellent safety profile regarding hepatic and metabolic parameters. Our comprehensive analysis revealed no clinically significant alterations in liver enzymes (ALT, AST) or markers of glucose and lipid metabolism (fasting glucose, TC, TG, HDL, LDL) among the treatment groups (Table 1). This metabolic neutrality is particularly reassuring, as it suggests quercetin neither induces liver toxicity nor disrupts normal lipid homeostasis - a critical consideration for chronic therapeutic use. The absence of adverse metabolic effects positions quercetin as a promising candidate for long-term management of hyperuricemia and its renal complications, especially in patients with concurrent metabolic disorders.

The renal damage caused by chronic hyperuricemia manifests through distinct pathological features - persistent inflammation, progressive fibrosis, and characteristic tissue remodeling - all visible through microscopic examination (Lu et al., 2024). Our histological assessment employed two complementary techniques: H&E staining to evaluate overall tissue integrity and Masson’s trichrome for specific collagen deposition patterns. Kidney sections from untreated hyperuricemic rats displayed the expected spectrum of damage, with pronounced inflammatory infiltrates, distorted glomerular architecture, and extensive fibrotic scarring throughout the tubulointerstitial compartments.

Quercetin intervention produced marked histological preservation at both dosage levels. The H&E slides showed noticeably fewer inflammatory foci and better-maintained renal structures, while trichrome staining revealed substantially less blue-stained collagen matrix (Figures 3A–C). This visual evidence of reduced fibrosis prompted us to investigate the molecular correlates. Through combined immunohistochemical and gene expression analyses, we quantified key fibrotic markers including collagen I, α-SMA, and fibronectin (Figures 3D–F). Quercetin treatment consistently suppressed expression of all three markers, demonstrating its capacity to interrupt the fibrotic cascade at multiple levels. These parallel findings - from macroscopic tissue preservation to molecular pathway modulation - paint a coherent picture of quercetin’s multifaceted renal protection. By simultaneously addressing both the inflammatory triggers and fibrotic consequences of hyperuricemia, the flavonoid appears to break the cycle of injury and maladaptive repair. The convergence of histological and biochemical evidence strongly supports quercetin’s potential as a disease-modifying agent in hyperuricemic nephropathy.

Our findings reveal one of quercetin’s most clinically relevant effects - its capacity to address the fundamental pathology of hyperuricemic nephropathy. The flavonoid demonstrated a dose-dependent ability to both minimize urate crystal deposition in renal tissue and enhance urinary uric acid elimination. This dual action was particularly evident at the 100 mg/kg dose, where histological analysis showed substantially fewer crystalline deposits compared to untreated controls (Figures 4A–C). What makes this observation particularly noteworthy is that these changes occurred alongside the compound’s other renoprotective effects, suggesting quercetin targets multiple pathological pathways simultaneously. Additionally, both 50 mg/kg and 100 mg/kg doses significantly increased urinary uric acid levels, with the high dose showing a more pronounced effect (Figure 4D), indicating enhanced renal uric acid clearance. To further investigate its regulatory effect on uric acid metabolism, immunohistochemical analysis revealed that quercetin downregulated the expression of renal GLUT9, a key uric acid reabsorption transporter. In contrast, URAT1 expression showed no significant difference among the groups (Figures 5A–D).

Crystal deposition contributes to renal inflammation, oxidative stress, and fibrosis, accelerating kidney injury. By limiting crystal accumulation, quercetin alleviates not only mechanical obstruction but also crystal-induced inflammatory responses. Although its renoprotective effects were less pronounced than those of febuxostat, quercetin still demonstrated significant efficacy, supporting its potential as an alternative or adjunctive agent for managing hyperuricemia.

One of the significant benefits of quercetin treatment in hyperuricemic rats was its ability to reduce renal inflammation (Kong et al., 2025). Hyperuricemia-induced inflammation plays a critical role in the progression of kidney damage, and quercetin demonstrated a potent ability to modulate these inflammatory responses. Immunohistochemistry demonstrated a substantial downregulation of NF-κB and TLR4, essential components in the orchestration of inflammatory signaling pathways (Figures 6A–C).

Moreover, quantitative PCR revealed a significant downregulation of IL-1β, MCP-1, and TNF-α transcripts following quercetin exposure (Figures 6D–F). Correspondingly, serum assays demonstrated a dose-responsive decrease in IL-1β, TNF-α, and IL-6 levels after treatment. (Figures 7A–C). These cytokines are pivotal mediators in immune cell recruitment and the amplification of renal inflammatory cascades. By suppressing their expression, quercetin effectively mitigates the sustained inflammatory milieu that underlies hyperuricemic nephropathy.

In hyperuricemia, excessive oxidative stress contributes to kidney damage by promoting inflammation, apoptosis, and fibrosis (Sui et al., 2024). The renal levels of key antioxidant enzymes—glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD)—were significantly elevated in rats receiving quercetin treatment (Figures 7D–G). This enzymatic enhancement indicates that quercetin contributes to strengthening the kidney’s antioxidant defense, which is typically compromised in the context of hyperuricemia.

Apoptosis, or programmed cell death, plays a pivotal role in the development of renal damage associated with hyperuricemic nephropathy (Liu et al., 2022b). To evaluate the effects of quercetin on this process, TUNEL staining was conducted and demonstrated a significant decrease in apoptotic cell numbers in the kidneys of rats treated with quercetin, compared to those in the model group (Figures 8A,B), implying a protective influence against renal cell loss. Consistent with this, Western blot analysis showed reduced expression of the pro-apoptotic marker Bax, along with elevated levels of the anti-apoptotic protein Bcl-2 following quercetin administration (Figures 8C–E). This shift in expression suggests that quercetin promotes cell survival by modulating apoptosis-related pathways, thereby mitigating renal injury.

ER stress has emerged as a crucial mechanism underlying kidney injury in hyperuricemia, driven by the accumulation of misfolded proteins and disruption of normal cellular homeostasis. (Andrade-Silva et al., 2025). Quercetin administration significantly mitigated ER stress in the kidneys of hyperuricemic rats, as shown by decreased immunohistochemical staining for key markers such as GRP78, phosphorylated PERK (P-PERK), and CHOP (Figures 9A–D). These observations were further confirmed by Western blot analysis, which showed a dose-dependent suppression of GRP78, P-PERK, IRE1α, ATF6, and CHOP—key mediators of the unfolded protein response (UPR) and ER stress signaling (Figures 10A–F). As activation of PERK, IRE1α, and ATF6 is typically induced by ER overload, their inhibition by quercetin suggests a reduction in cellular stress. By downregulating these pathways, quercetin may enhance protein folding capacity and promote renal cell survival, thereby mitigating ER stress–induced damage.

Male Sprague-Dawley rats weighing 180–220 g were randomly divided into five groups, with eight animals in each group: Control, Model, Quercetin-L (50 mg/kg), Quercetin-H (100 mg/kg), and Febuxostat (5 mg/kg). Hyperuricemia was induced in all groups, except the Control group, by daily oral administration of adenine (100 mg/kg, T0064, Targetmol, Shanghai, China) and potassium oxonate (1.5 g/kg, T5987, Targetmol, Shanghai, China) for 4 weeks. Concurrently, rats in the treatment groups received quercetin (50 mg/kg for Quercetin-L or 100 mg/kg for Quercetin-H, T2174, Targetmol, Shanghai, China) or febuxostat (5 mg/kg, T0773, Targetmol, China) by oral gavage, while the Control and Model groups received an equivalent volume of saline. After 4 weeks of treatment, the rats were anesthetized and then euthanized, with kidney tissues harvested for subsequent examination. All animal procedures were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Ethical Committee for Shanghai Pudong New Area People’s Hospital Shanghai.

Serum uric acid, creatinine, blood urea nitrogen, and other biochemical parameters were measured using an automatic biochemistry analyzer (Hitachi Model 7,600, Japan). Serum levels of tumor necrosis factor-α (TNF-α, ERC102a.96), interleukin-1β (IL-1β, ERC007.96), and interleukin-6 (IL-6, ERC003.96) were measured using ELISA kits from NeoBioscience (Shenzhen, China).

Systolic blood pressure was assessed with a non-invasive tail-cuff plethysmography device (ALC-NIBP, Shanghai, China). Prior to testing, rats were habituated to both the restrainer and the measurement equipment for a minimum of three consecutive days. Blood pressure readings were taken after the rats were placed in a temperature-controlled chamber. The tail was gently warmed, and three consecutive measurements were recorded. The average of these readings was used for statistical analysis to minimize variation. The procedure was performed at the same time each day to control for circadian effects.

Kidney tissues were fixed in 10% formalin and either embedded in paraffin or frozen in OCT compound. Tissue slices with a thickness of 4 μm were prepared for histological and immunohistochemical examination. General tissue structure was evaluated using hematoxylin and eosin (H&E) staining, whereas Masson’s trichrome staining served to evaluate renal fibrosis.

For immunohistochemical analysis, paraffin-embedded sections were first deparaffinized and then incubated with primary antibodies targeting α-SMA (Cat. No. 14395-1-AP, Proteintech, Wuhan, China), TLR4 (Cat. No. 66350-1-Ig, Proteintech, Wuhan, China), NF-κB (Cat. No. ET1603-12, HUABIO, Hangzhou, Zhejiang, China), GRP78 (Cat. No. HA722202, HUABIO, Hangzhou, China), phosphorylated PERK (p-PERK, Cat. No. PA5-102853, Thermo Fisher Scientific, United States), CHOP (Cat. No. 15204-1-AP, Proteintech, Wuhan, China),GLUT9(Cat No. 67530-1-Ig,Proteintech, Wuhan, China)and URAT1(Cat No. 14937-1-AP, Proteintech, Wuhan, China). After washing, sections were incubated with appropriate HRP-conjugated secondary antibodies. Immunoreactivity was visualized using diaminobenzidine (DAB) and counterstained with hematoxylin.

For birefringence analysis, frozen kidney sections were examined under a polarized light microscope to detect anisotropic uric acid crystals. Quantification of mean urate crystal area (MUC) was performed using ImageJ software.

Quantitative real-time PCR (qPCR) was conducted to assess the mRNA expression of inflammatory cytokines (IL-1β, MCP-1, TNF-α) and fibrosis-related genes (Collagen1, α-SMA, Fibronectin). Total RNA was extracted from kidney tissues using TRIzol reagent, and reverse transcription was performed according to the manufacturer’s protocol. The quality and quantity of RNA were assessed by a NanoDrop spectrophotometer (Thermo Fisher). cDNA synthesis was performed using a reverse transcription kit. The cDNA was then amplified using specific primers for each gene of interest, and the reaction was carried out on a real-time PCR system (Applied Biosystems StepOnePlus). The relative expression levels of target genes were normalized to GAPDH, and the fold change was calculated using the 2^-ΔΔCt method. All primers were designed based on published sequences (Peinnequin et al., 2004; Amanzada et al., 2014; He et al., 2017) and validated for specificity via BLAST analysis. The primer sequences and expected product sizes are as follows: Collagen1: forward 5′-ATC​CTG​CCG​ATG​TCG​CTA​T-3′, reverse 5′-CCA​CAA​GCG​TGC​TGT​AGG​T-3′ (207 bp); α-SMA: forward 5′-ATT​CCT​TCG​TGA​CTA​CTG​CTG​AG-3′, reverse 5′-CCC​ATC​AGG​CAG​TTC​GTA​GC-3′ (143 bp); Fibronectin: forward 5′-CAC​ACC​TGT​GAC​CAG​CAA​CAC-3′, reverse 5′-TCA​TCT​CCT​TCC​TCG​CTC​AGT​TC-3′; IL-1β: forward 5′-CAC​CTC​TCA​AGC​AGA​GCA​CAG-3′, reverse 5′-GGG​TTC​CAT​GGT​GAA​GTC​AAC-3′ (79 bp); MCP-1: forward 5′-AGG​CAG​ATG​CAG​TTA​ATG​CCC-3′, reverse 5′-ACA​CCT​GCT​GCT​GGT​GAT​TCT​C-3′ (107 bp); TNF-α: forward 5′-AAA​TGG​GCT​CCC​TCT​CAT​CAG​TTC-3′, reverse 5′-TCT​GCT​TGG​TGG​TTT​GCT​ACG​AC-3′ (111 bp); GAPDH forward 5′-GTA​TTG​GGC​GCC​TGG​TCA​CC-3′, reverse 5′-CGC​TCC​TGG​AAG​ATG​GTG​ATG​G-3′ (202 bp).

Kidney cortex samples were homogenized in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit (Pierce). Equal protein amounts (30 µg) were separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies targeting specific proteins. After washing, membranes were treated with HRP-conjugated secondary antibodies. Bands were detected using enhanced chemiluminescence (ECL), and densitometric quantification was carried out with ImageJ software. Protein levels were normalized against β-actin as a loading control.

The following primary antibodies were used for Western blot analyses: Bcl-2 (ET1603-11), Bax (ET1603-34), GRP78 (HA722202) IRE1α (HA723225), and ATF6 (HA601321) were purchased from HUABIO (Hangzhou, Zhejiang, China). Additional antibodies, including CHOP (15204-1-AP, WB) and β-actin (66009-1-Ig, WB), were obtained from Proteintech (Wuhan, Hubei, China). Phosphorylated PERK (P-PERK, 3,179) was purchased from Cell Signaling Technology (CST, Boston, United States).

Oxidative stress-related enzyme levels in tissue were quantified using ELISA kits obtained from NanJing JianCheng Bioengineering Institute (Nanjing, Jiangsu, China). The enzymes measured included glutathione (GSH, Cat. No. A006-2-1), glutathione peroxidase (GSH-Px, Cat. No. A005-1-2), catalase (CAT, Cat. No. A007-1-1), and superoxide dismutase (SOD, Cat. No. A001-3-2).

TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was performed to detect apoptotic cells in kidney tissue sections. Kidney tissues were fixed in paraformaldehyde, embedded in paraffin, and sectioned into 4-μm-thick slices. Apoptotic cells were detected using a commercial TUNEL detection kit (T6014S, Uelany, Suzhou, Jiangsu, China), following the manufacturer’s instructions. After incubation with the TUNEL reagent, the sections were counterstained with DAPI for nuclear visualization. The number of TUNEL-positive cells was counted under a fluorescence microscope.

Data are shown as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for statistical comparisons, followed by Tukey’s post hoc test for multiple group comparisons. A p-value below 0.05 was regarded as statistically significant. Graphs were created with GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA).