Renoprotective effects of perennial ryegrass attenuate UA mediated renal damage via modulating gut microbiota and anti-oxidative defense

Abstract

Hyperuricemia (HUA) is a metabolic disorder and a major cause of gout in geese, commonly exacerbated by concentrate diets rich in protein and calcium. Elevated uric acid (UA), a potent pro-oxidant, contributes to renal injury and is strongly influenced by dietary composition and gut microbiota. Therefore, research for natural feed ingredients that can regulate UA homeostasis and oxidative stress is particularly essential. Perennial ryegrass, a fiber-rich forage containing abundant minerals and antioxidant flavonoids, may mitigate these adverse effects, however its renoprotective potential in goslings remains unclear. This study investigated the protective effects of perennial ryegrass against UA-induced oxidative stress and renal damage in goslings. High-through 16S rRNA and LC-MS metabolomics were performed to determine the effect of ryegrass on the gut microbiota-metabolite axis in goslings. Biochemical indexes, histopathology, immunofluorescence, gene expressions, and western blotting were used to observe the renoprotective potential of ryegrass. Ryegrass significantly lowered serum UA and creatinine levels (p < 0.05) by upregulating renal UA-excreting transporters (ABCG2, OAT1 and OAT3) and downregulating UA-reabsorbing transporters (URAT1 and GLUT9). Histopathology and microbial profiling revealed that ryegrass reduced tubular damage, fibrosis, and immune cell infiltration, accompanied by enrichment of short chain fatty acid-producing bacterial taxa. Transcriptional, protein, and immunofluorescence analysis demonstrated that ryegrass suppressed inflammatory cytokines (IL-1β, IL-18, TNF-α) through inhibition of the NLRP3/caspase-1/Keap1 axis, likely related to decreased renal UA burden. In parallel, it strengthened antioxidant defenses by upregulating Nrf2, which increased GSH-Px, CAT, and SOD activities and lowered MDA and ROS accumulation. Metabolomics further demonstrated higher levels of antioxidant metabolites (caffeic acid, kaempferol, skimmin, quercetin, and ferulic acid) in ryegrass-fed goslings relative to the concentrate-fed group. These findings highlight the renoprotective potential of perennial ryegrass in alleviating UA-induced oxidative renal injury and provide new insights into its value as a functional forage in the geese industry.

Introduction

The livestock industry increasingly prioritizes animal health and welfare, particularly in poultry production, where birds are frequently exposed to environmental and nutritional stressors (Son et al., 2022). Diets rich in protein, calcium and fat can disrupt metabolic homeostasis and significantly elevate UA levels, leading to HUA and oxidative damage (Mehmood et al., 2019; Zhang et al., 2020; Zhao et al., 2022). Elevated UA, acting as a potent intracellular pro-oxidant, induces lipid peroxidation, mitochondrial dysfunction, and apoptosis in vital organs such as the kidney, liver, and pancreas (Gu et al., 2008; Gu and Xu, 2010; Kołodziej et al., 2017). UA homeostasis is regulated by hepatic production and excreted by kidney and intestine, which together remove nearly all systemic UA (Yang et al., 2024). The balance between UA reabsorption and secretion depends on the coordinated activity of transporters including GLUT9, URAT1, OAT1, OAT3, and the ATP-binding cassette transporter ABCG2 (Nigam and Bhatnagar, 2018). Disruption of these pathways contributes to UA accumulation resulting oxidative stress and inflammatory responses, mainly cause renal injury.

Oxidative stress defined as an imbalance between reactive oxygen species (ROS) generation and antioxidant defenses, plays a central role in UA-mediated renal pathology (Zhang et al., 2019). Excess ROS activates the NOD-like receptor protein 3 (NLRP3) inflammasome, triggering caspase-1 activation and maturation of pro-inflammatory cytokines IL-1β, IL-18 and TNF-α (Hseu et al., 2022; Swanson et al., 2019; Tschopp and Schroder, 2010). Under oxidative stress, Keap1 dissociates from nuclear factor erythroid 2 related factor 2 (Nrf2), which upregulates antioxidant defense by enabling the transcription of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) (Dai et al., 2019; Cao et al., 2021; Hseu et al., 2022). Persistent UA elevation and ROS accumulation result in glomerulosclerosis, tubulointerstitial fibrosis, and progressive renal dysfunction (Keenan, 2020; Piani and Johnson, 2021). Therefore, strategies that lower UA levels and attenuate oxidative renal injury are essential for maintaining kidney health (Yang et al., 2010).

In poultry, the first few weeks post-hatch are critical for survival, immune maturation, and organ development (Moreira Filho et al., 2019; Ravindran and Abdollahi, 2021). Among poultry, geese are particularly susceptible to HUA and gout between 1 and 20 days of age, leading to high mortality and substantial economic losses (An et al., 2020; Zhang et al., 2018). In China, where 500–600 million geese are produced annually, more than 50% may experience gout during the growing period (Wang et al., 2021). Current pharmacological treatments for gout, including steroidal and non-steroidal anti-inflammatory drugs, are limited by adverse effects such as gastric irritation, nephrotoxicity, and cardiovascular risk, underscoring the need for safe, natural alternatives (Xu et al., 2021). Natural plant-derived compounds with antioxidant and anti-inflammatory potential effectively preventing many diseases in animals (Bederska-Łojewska et al., 2017; El-Wahab et al., 2022; Silva et al., 2005).

Perennial ryegrass (family Poaceae) is a nutrient-dense forage rich in digestible fiber, essential minerals, and bioactive molecules including alkaloids, flavonoids, glycosides, isoflavonoids, and polyphenols (Chilibroste et al., 2000). Recent studies suggest that perennial ryegrass can modulate gut microbiota and reduce inflammatory cytokines, thereby protecting against oxidative injury (Ali et al., 2022; Choi et al., 2017; Zhao et al., 2022; Zheng et al., 2021). Some other crops such as cereal rye from same family (Poaceae) were also reported for their ethnomedical properties and used as treatment for urinary problems (Csikós et al., 2021). As herbivores, geese efficiently utilize plant-derived fiber, which promotes beneficial bacterial genera such as Faecalibacterium, Ruminococcus, Butyricicoccus, and Akkermansia while suppressing opportunistic taxa (Chen et al., 2018; Kim et al., 2018). Although traditional goose production relied on grazing, this is impractical in intensive systems; thus, integrating forage such as ryegrass into commercial diets offers a cost-effective and sustainable nutritional strategy (Wood et al., 2011).

Recent studies have highlighted the anti-inflammatory properties of ryegrass, but its renoprotective mechanisms remain unclear and require a comprehensive investigative approach. In this study, we investigated the renoprotective effects of perennial ryegrass in goslings subjected to UA-induced oxidative stress. We evaluated changes in microbiota-metabolite axis, biochemical markers, renal structure, systemic inflammatory responses, and key signaling Nrf2/NLRP3 pathways. This comprehensive approach described how dietary inclusion of ryegrass modulate gut microbiota-metabolites axis and alleviate UA-induced oxidative renal injury and promote geese health.

Material and method

The research bioethics Committee of Henan Agricultural University approved all the protocols for in-vivo experiments (Approval No. HENAU-202305). The Animal Ethics Committee of the College of Animal Science at HENAU provided ethical clearance for animal housing.

Experimental design

A group of 300 post hatched goslings (Wanpu) were purchased from Henan Daidai Goose Agriculture and Animal Husbandry Development Co., Ltd (Zhumadian, China). Goslings with average body weight (90 ± 5 g) were allocated into two experimental groups for 3-week feeding period: (1) concentrate feed consuming group (CF, n = 150) and (2) concentrate feed mixed with chopped perennial ryegrass (70:30) consuming group (PRD, n = 150) with 6 replications having 25 goslings in each. Each replicate (pen of 25 geese) was considered the experimental unit. For statistical analysis, measurements from individual geese within each replicate were averaged to obtain one mean value per replicate, and treatment effects were considered significant at P < 0.05. Chopped perennial ryegrass was added to the concentrate diet of goslings as a source of plant based high fiber and phytochemical compounds. The ratio (70:30) was selected based on our previous experiment to balance nutritional intake and fiber supplementation. Goslings were raised in semi-controlled shed with plastic net floor, fiberglass feeders and automatic bell drinkers with continuous supply of water and physical parameters such as light and temperature were maintained according to company guidelines. Prior to the experiment, the indoor environment was disinfected, and daily management was done according to management regulations of the animal ethics committee of College of Animal Science HENAU. For first 7 days, goslings were provided ad libitum feed for whole day. Feed residues were collected and weighed the next morning to calculate daily feed intake. From day 8 to 21, goslings were fed in restricted manner, three times every day (after 8 hours). Residual feed was collected and weighed after two hours of feeding to determine feed intake. During the 21-d trial, the concentrate feed (CF) was formulated with maize (57.8%), soybean meal (30%), wheat bran (5.0%), sunflower meal (2.0%), rice husk (1.3%), limestone (1.1%), dicalcium phosphate (1.4%), lysine (0.04%), DL-methionine (0.1%), premix (1.0%), and salt (0.3%). The diet was formulated to provide 11.4 MJ/kg of metabolizable energy and 19.57% crude protein. The calculated crude fiber, calcium, total phosphorus, lysine, and methionine contents were 4.50%, 0.86%, 0.36%, 0.92%, and 0.37%, respectively. The detailed ingredient and vitamin-mineral premix composition is presented in (Supplementary Table 1). Gosling’s physical parameters, mortality and feed intake were recorded daily to measure average daily feed intake (ADFI). Average daily gain (ADG) and feed conversion ratio (FCR) were recorded weekly. The nutritional profile and bioactive polyphenolic compound composition of perennial ryegrass are presented in Supplementary Table 2. The nutritional composition of both groups was given in Table 1.

Table 1.

Nutrients composition of diets.

Feed components	Concentrate Feed	Commercial diet: Perennial ryegrass (70:30)
CP (%)	19.57	18.97
ME (MJ/Kg)	11.4	10.83
CF (%)	4.5	11.1
Neutral detergent fibre (%)	11.72	19
Acid detergent fibre (%)	5.55	10.5
Ash (%)	6.34	7.1
Calcium (%)	0.85	0.86
Total Phosphorus (%)	0.36	0.39

Crude Protein= CP, Metabolizable energy= ME, Crude Fiber= CF.

Sample collection

Four goslings (per replicate) were randomly selected followed by 12 hours fasting. Blood was drawn by ulnar vein and placed in serum separator labeled tubes and then centrifuged for 10 min at 3000 × g, held at −20°C for further assay. Cervical dislocation method used for collection of fresh cecal chyme using centrifuge tubes (5 ml), held at −80°C for later use. Additionally, the tissue samples from ileum, cecum and kidney were fixed by using immersion (4%) and neutral buffered formalin (10%).

Biochemical parameters

Serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), and total cholesterol (T-Chol) were determined using commercial assay kits. The urate transporters ABCG2, OAT1, OAT3, GLUT9, and URAT1 were quantified by ELISA. Serum aliquots and renal tissues were used to assess pro-inflammatory cytokines and mediators, including interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂), as well as anti-inflammatory cytokines IL-10 and IL-4. Serum and renal oxidative markers malondialdehyde (MDA), reactive oxygen species (ROS), and nitric oxide (NO) along with antioxidant enzymes glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were also analyzed using ELISA kits. All measurements were performed in triplicate for each sample. Catalog numbers of the specific ELISA kits are provided in Supplementary Table 3.

Renal histological examination

Kidney tissues were fixed in 10% paraformaldehyde for 24 h at room temperature. Following fixation, tissues were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Paraffin blocks were sectioned at 5 µm thickness. The sections were deparaffinized, rehydrated, and stained with Harris’ hematoxylin solution for 6 h at 60-70°C, followed by rinsing under running tap water until the water became clear. Subsequently, tissue differentiation was performed using 10% acetic acid and 85% ethanol for 2 h and 10 h respectively, followed by thorough washing with tap water. The sections were immersed in a saturated lithium carbonate solution for 12 h and then rinsed again in tap water. Finally, counterstaining was performed with eosin Y ethanol solution for 48 h, after which sections were dehydrated, cleared, and mounted for microscopic examination (Ali et al., 2022). Required indices were inspected by employing Image-Pro Plus 6.0 software. The renal histopathological score was determined according to the method described by (Li et al., 2022).

Gene expression

Total RNA was isolated from kidney tissue using the Animal Tissue RNA Isolation Kit and RNA integrity and concentration were determined via NanoDrop spectrophotometry. The reverse transcription Master Mix used to generate cDNA from RNA (500 ng). Quantitative real-time PCR was conducted on the LightCycler 480 II platform employing the Power SYBR Green Master Mix. Each 10 µL reaction comprised 5 µL of SYBR Green mix, 1 µL of cDNA, 0.5 µL each of forward and reverse primers (10 µM), and 3 µL of nuclease-free water. GAPDH was used as the internal reference gene, and relative transcript levels were calculated using the 2^-ΔΔCt method. Specific primer sequences used in the study are listed in Supplementary Table 4.

Western Blot

Kidney tissues (three samples/group) proteins were extracted by homogenization in RIPA buffer with protease and phosphatase inhibitors and total protein concentration was determined. Protein (40 μg per sample) was subjected to separation via SDS-PAGE and transferred onto PVDF membranes. Membranes were subsequently blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies. Protein bands were detected using a FluorChem R imaging system and quantified using ImageJ software. Specific kilodaltons (KDa) for proteins representing in Supplementary Table 5.

Immunofluorescence analysis

Kidney tissue sections were fixed in 4% paraformaldehyde for 10 min, dewaxed in xylene for 15 min, and rehydrated through graded ethanol to distilled water. Antigen retrieval was carried out in citrate buffer (pH 6.0) using a pressure cooker; after reaching full pressure, tissues were heated for 2 min and cooled naturally to room temperature. For low-abundance proteins, EDTA buffer (pH 9.0) was alternatively employed. Following antigen retrieval, sections were blocked with 10% fetal bovine serum (FBS) for 30 min at room temperature to prevent nonspecific binding. The slides were then incubated overnight at 4°C with primary antibodies against NLRP3 (AF06824, 1:1000) and Nrf2 (AF300189, 1:2000). After washing with PBS, sections were incubated for 50 min at room temperature in the dark with HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (1:400). Fluorescent signal amplification was performed using a tyramide signal amplification kit containing tyramine sodium fluorescein (Servicebio, G2150-1 L) in PBST with 0.0003% H₂O₂ for 20 min at room temperature. Nuclei were counterstained with DAPI for 5-10 min at room temperature and mounted with antifade medium (Servicebio, G1401, Fluoroshield with DAPI). Slides were visualized under a Leica TCS SP8 STED confocal microscope (20 × objective). Fluorescein signals were excited with a 488 nm laser and detected using a 520 nm emission window, while DAPI was excited at 405 nm. Fluorescence intensity was analyzed using ImageJ 6.0 (NIH, USA) by converting fluorescence images to grayscale. Integrated density (IntDen) and positive signal area were measured, and average fluorescence intensity was calculated as IntDen/Area. Quantification was performed from three randomly selected fields per sample. Negative controls were prepared in parallel by omitting the primary antibody to assess nonspecific binding and background fluorescence.

Gut microbiota analysis

Genomic DNA was isolated from cecal content using the PSP Spin Stool DNA Plus Kit, following the manufacturer’s protocol. The integrity and quality of the extracted DNA were verified prior to amplification of the 16S rRNA gene, targeting the V3-V4 hypervariable regions using the primer pair F515/R806. Microbial diversity analysis was determined as described by (Zulfiqar et al., 2025) .

Serum metabolomics analysis

A serum sample of 100 μL was mixed with 400 μL of a methanol:acetonitrile solution (1:1) and subjected to ultrasonication for 30 minutes. The mixture was then refrigerated for 30 minutes at −20°C and centrifuged for 10 minutes (1,200 rpm, 4°C). The supernatant evaporated under vacuum and resuspended in a solution of 50% methanol containing 5 ppm of 2-chlorophenylalanine. After a second centrifugation, the filtrate was transferred into sample vials for liquid chromatography-mass spectrometry (LC-MS). To verify the accuracy and experimental reliability, quality control (QC) samples were prepared by 10 μL from each sample. Gradient elution method was used for Chromatography on ACQUITY UPLC HSS T3 column (flow rate 0.4 mL/min, 40°C). The mobile phases consisted of A (0.1% formic acid in water) and B (acetonitrile with 0.1% formic acid). Data from Mass spectrometry were acquired and processed by using Thermo Orbitrap Exploris 120 (both positive and negative ion modes) and Compound Discoverer™ 3.3 respectively. The peaks area was normalized by removing peaks having a coefficient of variance greater than 30%. Metabolites were identified using a custom-built PSNGM database, along with spectral libraries (mzCloud, LIPID MAPS, HMDB, MoNA, and NIST_2020_MSMS). Differentially abundant metabolites were subjected for Cluster analysis using the Pheatmap package in R (v1.0.12). Furthermore, the Ropls package in R was used for Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). To avoid overfitting, Permutation test was performed. Differentially abundant metabolites in groups were characterized based on the criteria of P < 0.05, FC > 1 or < 1, and VIP > 1, with VIP values calculated from the OPLS-DA results.

Statistical analysis

Data analysis was performed using IBM SPSS Statistics version 26. All data were expressed as mean ± standard deviation (SD). Since the experiment involved two dietary groups, an unpaired Student’s t-test was used to determine differences between groups. Prior to analysis, the assumptions of normality and homogeneity of variance were verified using the Shapiro-Wilk and Levene’s tests, respectively. Results with P < 0.05 were considered statistically significant. Pearson’s correlation analysis for host biomarker associations was carried out using OECloud tools (https://cloud.oebiotech.cn).

Results

Physiological and biochemical indexes

The two experimental groups showed no significant difference in ADFI during the first two weeks. However, there was significant increase in ADFI at 3rd week in PRD group (P < 0.05). The average daily gain (ADG) and FCR were improved (P < 0.05) in CF compared to PRD group at 2nd and 3rd week (Table 2). Serum metabolic profile (Fig. 1A1-A5) was significantly influenced in both groups. The UA level was significantly (P < 0.05) elevated in CF group at 2nd and 3rd week. Meanwhile, serum Cr and BUN were also showed increased levels (P < 0.05) in CF group as compared with PRD. Total protein and cholesterol levels were observed lower in PRD group.

Table 2.

Weekly growth performance.

Parameters	CF			PRD			p-value
	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3	Week 1	Week 2	Week 3
ADFI (g)	81.2 ± 1.15	128.6 ± 5.15	174±6.5	82 ± 2.58	130±6.10	179.3 ± 5	>0.05	>0.05	>0.05
ADG (g)	45.7 ± 1.80	48.3 ± 2.01	95.5 ± 3.50 a	44.8 ± 2.7	47.2 ± 2.03	86.5 ± 4.06 b	>0.05	>0.05	<0.05
FCR	1.8 ± 0.17	2.66± 0.08	1.82±0.09 b	1.83 ± 0.02	2.75±0.06	2.07 ± 0.07 a	>0.05	>0.05	<0.05
Mortality Rate	3.3 ± 0.50 a	3.33 ± 0.58 a	3 ± 1 a	0.3 ± 0.5b	1.33±0.5 b	0.67 ± 0.58 b	<0.05	<0.05	<0.05

a, b, c, d showed in the same row with different letters differ significantly at P < 0.05.

CF, Concentrate feed group; PRD, Perennial ryegrass included group.

Abbreviations: ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio.

Fig. 1.

Renal basic functioning parameters and renal uric acid transporters. (A1-A5) Renal basic functioning parameters (serum levels), uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), total cholesterol level (T-Chol), (B1-B3) Serum level of UA excreting transporters, organic anion transporter 1 and 3 (OAT1, OAT3), ATP-binding cassette subfamily G member 2 (ABCG2) respectively, (B4-B5) UA reabsorbing transporters, glucose transporter 9 (GLUT9), urate transporter 1 (URAT1) respectively, (C) transcriptional abundance levels of OAT1,OAT3 and ABCG2, (D) Western blot representation of OAT1,OAT3 and ABCG2, (E) Transcriptional abundance levels of GLUT9 and URAT1, (F) Western blot representation of URAT1 and GLUT9. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Uric acid excreting and reabsorbing transporters

Perennial ryegrass significantly enhanced renal uric acid excretion by upregulating key UA-excreting transporters in young goslings (Fig. 1B1–B3). Serum levels of the excretory transporters OAT1, OAT3, and ABCG2 were significantly higher in the PRD group compared with the CF group (P < 0.05). In contrast, the UA-reabsorbing transporters GLUT9 and URAT1 were elevated in the CF group (Fig. 1B4–B5). Consistent with these findings, renal mRNA abundance and protein expression of UA transporters demonstrated a similar pattern (Fig. 1C–F). The PRD group exhibited significantly increased transcriptional and protein levels of OAT1, OAT3, and ABCG2 (P < 0.05), whereas GLUT9 and URAT1 were expressed at higher levels in the CF group. Dietary perennial ryegrass effectively suppressed the expression of UA-reabsorbing transporters and promoted the expression of excretory transporters, collectively supporting its renoprotective and uricosuric effects.

Histopathological changes in the kidneys

Histopathological examination revealed clear protective effects of perennial ryegrass on renal structure (Fig. 2A). H&E staining showed that goslings in the PRD group maintained normal glomerular and tubular architecture, whereas the CF group exhibited extensive lesions characterized by tubular dilation, epithelial cell necrosis, inflammatory cell infiltration, and early fibrotic deposition. Consistently, the kidney coefficient was lower in the PRD group, while the glomerular tuft area was significantly increased in the CF group (P < 0.05), corresponding to a higher overall kidney injury score. Biochemical indicators of renal inflammation further supported these findings (Fig. 2B). Renal concentrations of COX-2 and PGE2 were significantly increased in the CF group, whereas NO levels were reduced at week 3, indicating enhanced inflammatory activity in concentrate-fed goslings. Molecular analysis aligned with the histological and biochemical results (Fig. 2C). The transcriptional levels of renal injury biomarkers, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were significantly higher in CF group, while ryegrass significantly increased the Klotho expression indicating reduced renal damage. Additionally, key fibrotic markers including collagen-1, TGF-β1, and fibronectin were significantly reduced by inclusion of perennial ryegrass compared with CF (Fig. 2D). Ryegrass effectively attenuates renal inflammation, injury, and fibrosis, thereby preserving kidney structure and function in young goslings.

Fig. 2.

Renal histological analysis. (A) H&E representative images of kidney tissue and their relative indices, scale bar = 200 μm. (B) Renal inflammatory mediators; cyclooxygenase-2 (COX-2), Prostaglandin E2 (PGE2), and nitric oxide (NO), (C) Transcriptional abundance levels of early renal injury biomarkers, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and Klotho, collagen-1, Transforming Growth Factor Beta 1 (TGF-β1), and Fibronectin. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Immunity status

The serum and renal inflammatory cytokines (IL-1β, IL-18 and TNF-α) were significantly increased (P < 0.05) in CF group, indicating pronounced UA-induced inflammatory activation. Whereas, perennial ryegrass significantly increased the serum and renal anti-inflammatory cytokines (IL-10 and IL-4) PRD group, demonstrating the immunomodulatory effects (Fig. 3A-D). Concentrate diet significantly increased the serum and renal Oxidative mediators ROS and MDA (P < 0.05), while ryegrass significantly increased the serum and renal antioxidant enzymes CAT, SOD and GSH-PX activity in PRD group (Fig. 3E-H). We observed the similar trend in term of renal transcriptional abundances (Fig. 3I). These results indicate that ryegrass effectively mitigates UA-induced oxidative and inflammatory injury while enhancing systemic and renal antioxidant defenses.

Fig. 3.

Immunity status. (A) Serum pro-inflammatory cytokines, interleukin-1β (IL-1β), interleukin-18 (IL-18), and tumor necrosis factor- α (TNF-α), (B) Serum anti-inflammatory cytokines, interleukin-10 (IL-10, and interleukin-4 (IL-4), (C-D) Renal pro-inflammatory and anti-inflammatory cytokines, (E) Serum oxidative mediators, reactive oxygen species (ROS), and malondialdehyde (MDA), (F)) Serum antioxidant enzymes, glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD), (G-H) Renal oxidative mediators and antioxidant enzymes, (I) Renal transcriptional abundances of pro-inflammatory, anti-inflammatory cytokines, oxidative mediators and antioxidant enzymes. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Renal NLRP3/Nrf2 signaling mechanism

Perennial ryegrass effectively suppressed the inflammatory signaling pathway (Fig. 4A–B). The transcriptional and protein levels of NLRP3, caspase-1, and Keap1 were significantly reduced in the PRD group, indicating effective suppression of NLRP3 inflammasome activation and oxidative stress signaling. In contrast, Nrf2 expression was significantly upregulated at both mRNA and protein levels in the PRD group compared with CF. Immunofluorescence staining further supported these findings (Fig. 4C–D). The ryegrass exhibited clear downregulation of NLRP3 expression and upregulated Nrf2 localization within renal tissues, consistent with reduced inflammation and strengthened antioxidant responses. These data demonstrate that perennial ryegrass activates Nrf2-driven antioxidant defenses while inhibiting NLRP3 inflammasome activation, thereby mitigating UA-induced inflammatory and oxidative injury in the kidney.

Fig. 4.

NLRP3/Nrf2 pathway. (A) Transcriptional abundance levels of, NOD-like receptor protein 3 (NLRP3), Caspase-1, Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2), (B) Protein abundance levels of NLRP3, Keap1 and Nrf2, (C) Representative images of immunofluorescence staining of kidney tissue for NLRP3. Fluorescence intensity was quantified using ImageJ 6.0. Blue: nucleus (DAPI); Red: NLRP3 staining; Merge; combination of blue and red indicating nuclear translocation of NLRP3, scale bar = 200 μm. (D) Representative images of immunofluorescence staining of kidney tissue for Nrf2 K-viewer software. Fluorescence intensity was quantified using ImageJ 6.0. Blue: nucleus (DAPI); Red: Nrf2 staining; Merge; combination of blue and red indicating nuclear translocation of Nrf2, scale bar = 200 μm. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Gut microbial composition

Microbial diversity analysis demonstrated that perennial ryegrass significantly improved gut microbial composition by increasing microbial richness (Sob index) and diversity (Simpson index) compared with the CF group (Fig. 5A-B). At beta diversity level, PCoA analysis described the distinct bacterial distribution between both groups (Fig. 5C). At ASV level, Venn diagram described that perennial ryegrass increased the bacterial abundance in PRD group (common = 57, unique = 2551), compared with CF (common = 57, unique = 521) (Fig. 5D). Community pie plots showed the difference at phylum level, perennial ryegrass significantly increased the Bacteriodota and Verrucomicrobiota phyla, while Firmicutes abundance decreased in PRD group (Fig. 5E). At genus level, bar plots showed the distribution of bacterial genus between groups (Fig. 5F). Wilcoxon rank-sum test described that perennial ryegrass increased the beneficial short chain fatty acid producing genra including Akkermansia, Shuttleworthia, Oscillibacter, Desulfovibrio, Lachnoclostridium, NK4A214 and butyricicoccus, while cholesterol producing Turicibacter and Megamonas were abundant in CF group (Fig. 5G). Network analysis showed distinct clustering of microbial communities by dietary group, with beneficial probiotic species strongly associated with the PRD group, indicating a diet-driven restructuring of the microbial ecosystem (Fig. 5H). These findings demonstrate that perennial ryegrass significantly enhances gut microbial diversity and promotes the proliferation of health-associated bacterial species, contributing to improved gut and systemic health in goslings.

Fig. 5.

Gut microbiota status. (A-B) Alpha diversity indexes at ASV level; Sobs and Simpson indexes, (C) Beta diversity index at ASV level; PCoA, (D) Venn diagram at ASV level describing common and unique number of bacteria between groups, (E) Community Pie plot representation of bacteria at phylum level, (F) Community bar plot representation of bacteria at genus level, (G) Wilcoxon rank-sum test plot for differentially abundant genera between both groups, (H) networking analysis between experimental group and expressed species.

Metabolomics

Perennial ryegrass effectively modulated microbial-metabolite axis. Venn diagram analysis showed that the PRD group possessed a greater number of unique metabolites (51, 4.10%) compared with the CF group (24, 1.93%), indicating enhanced metabolic diversity in response to ryegrass inclusion (Fig. 6A). KEGG pathway enrichment analysis demonstrated that amino acid-related metabolic pathways were significantly upregulated in the PRD group (Fig. 6B). Notably, lysine biosynthesis, nucleotide metabolism, ABC transporters, tryptophan metabolism, and arginine proline metabolism were among the most enriched pathways. Classification of metabolite categories further showed that alkaloids, terpenoids, phenylpropanoids, and flavonoids were the dominant groups in the PRD group, reflecting an enhanced antioxidant metabolic profile (Fig. 6C). VIP and heatmap analysis identified clear differences in metabolite expression between groups (Fig. 6D). Several antioxidant metabolites including caffeic acid, kaempferol, skimmin, quercetin, ferulic acid, quinic acid, l-tryptophan, l-proline, and capillarisin were significantly enriched in the PRD group (Fig. 6E). These compounds are well known for their free radical-scavenging, anti-inflammatory, and cytoprotective functions. Spearman correlation analysis further demonstrated that these antioxidant metabolites exhibited strong positive correlations with anti-inflammatory cytokines (IL-10, IL-4) and antioxidant enzymes (GSH-Px, CAT, SOD), while showing negative correlations with inflammatory cytokines (IL-1β, TNF-α) and oxidative mediators (ROS, MDA) (Fig. 6F). These findings indicate that perennial ryegrass enhances systemic antioxidant defense by promoting the production of bioactive antioxidant metabolites, which in turn support anti-inflammatory and redox-regulating pathways in goslings.

Fig. 6.

Differentially expressed metabolites and antioxidant metabolites. (A) Venn plot diagram (B) KEGG pathways expression between groups, (C) abundance of differentially expressed metabolites in different catagories, (D) VIP diagram, (E) abundance levels of antioxidant metabolites between experimental groups, (F) correlation analysis between antioxidant metabolite and immunity indices. The asterisks symbol indicates significant differences *P < 0.05, **P < 0.01.

Discussion

With increasing living standards and excessive protein intake, dietary patterns have shifted toward higher purine loads, contributing to the global rise in HUA and gout (Hyndman et al., 2016). Goslings are particularly vulnerable, with mortality rates reaching 70% in severe cases and exhibiting pathophysiological characteristics comparable to human gout (Fu et al., 2024). Dietary interventions using plant-based fibers and phytochemicals have gained attention due to their ability to modulate UA transporters and mitigate oxidative stress (Bian et al., 2018; Nigam and Bhatnagar, 2018; Zheng et al., 2021). In this context, our study demonstrates that dietary inclusion of perennial ryegrass exerts protective effects against HUA-associated renal dysfunction in young goslings. By integrating physiological, biochemical, histopathological, immunological, microbial, and metabolomic evidence, our findings highlight perennial ryegrass as a potent modulator of urate handling, oxidative stress and the gut-kidney metabolic axis. The results collectively establish ryegrass as a natural dietary strategy capable of attenuating UA-induced renal injury through coordinated microbiota-metabolite interactions (Fig. 7).

Fig. 7.

Renoprotective effects of perennial ryegrass. Perennial ryegrass included diet (PRD) enhanced the UA excretion through upregulating UA excreting transporters (ABCG2, OAT1 and OAT3) and downregulating the UA reabsorbing transporters (Glut9 and URAT-1). PRD increased the production of gut probiotic bacteria and bacterial mediated antioxidant metabolites. PRD inceeased the activity of antioxidants enzymes (GSH-PX, CAT and SOD) and anti-inflammatory cytokines (IL_4 and IL-10) also increased. PRD effectively reduced the UA level, subsequently decreased the oxidative mediators (ROS and MDA), renal injury biomarkers (COX-2, PGE2, KIM-1 and NGAL) and inflammatory cytokines (IL-1β, IL-18 and TNF-α) to protect the renal tissues. PRD upregulated the antioxidant defense by suppressing the expression of NLRP3, caspase-1 activity and activating Nrf2. Collectively, PRD protects renal tissues by modulating microbial communities, upregulating antioxidant defense and reducing UA mediated oxidative stress and inflammation.

Although ADG and FCR were higher in the CF group during the later period, these improvements likely reflect excessive protein and energy intake associated with concentrate feeding rather than improved health status. The reduced ADG observed with perennial ryegrass aligns with previous studies reporting that pasture-fed geese show lower carcass fat and eviscerated yield but healthier growth and reduced mortality with economic benefits (Ali et al., 2022; Sekh and Karki, 2022; Song et al., 2017). Importantly, the elevated UA levels in CF-fed goslings confirm that high-concentrate diets impose a metabolic burden leading to impaired UA excretion. Ryegrass supplementation effectively promoted uricosuric activity, as evidenced by increased expression of OAT1, OAT3, and ABCG2 and suppression of the reabsorptive transporters GLUT9 and URAT1 (Chen et al., 2015; Lee et al., 2017; Perez-Ruiz et al., 2015). A number of studies also have been confirmed that the several plants (stevia, baicalein and chicory) influenced the expressions of these transporters (Bian et al., 2020; Zheng et al., 2021). The reduced serum UA, creatinine, and BUN levels in the PRD group strongly indicate enhanced renal clearance and preserved kidney filtration capacity, consistent with findings from (Desai et al., 2017; Fu et al., 2024; Wang et al., 2011; Xi et al., 2022).

Histopathological assessments further confirmed the renoprotective effects of ryegrass. The CF group exhibited primary features of UA-induced nephropathy, including tubular dilation, necrosis, inflammatory infiltration, and early fibrotic deposition, all of which are characteristics of renal oxidative and inflammatory stress (Gherghina et al., 2022; Su et al., 2020; Sun et al., 2021). The reduced kidney coefficient and preserved glomerular architecture in the PRD group reflect significantly reduced tissue damage. Molecular biomarkers highlighted these findings as elevated KIM-1, NGAL, COX-2, and PGE2 in the CF group indicate active renal injury and inflammation (Enomoto et al., 2002; Yang et al., 2019, 2024). Whereas ryegrass significantly reduced these indicators while increasing the renoprotective gene Klotho. Downregulation of key fibrotic mediators (TGF-β1, collagen-1, and fibronectin) further suggests that ryegrass inhibits the progression toward renal fibrosis. These data indicate that ryegrass maintains renal integrity by suppressing injury and fibrotic pathways commonly triggered by HUA.

Uric acid acts as a pro-oxidant induces oxidative stress and activates the NLRP3 inflammasome that triggers inflammatory signaling mechanism. The CF group exhibited elevated IL-1β, IL-18 and TNF-α, production in response to UA accumulation (Donovan et al., 2020; Jo et al., 2016). Whereas perennial ryegrass inhibited these inflammatory cytokines release by suppression of NLRP3 inflammasome signaling, suggesting immune homeostasis (Mangan et al., 2018). The CF group increased renal expression of NLRP3, caspase-1, and Keap1, consistent with inflammasome activation and oxidative stress. In contrast, ryegrass downregulated these inflammatory mediators while upregulating activation of Nrf2 signaling as pivotal antioxidant pathway underlying the renoprotective effects of ryegrass. Nrf2 activation promotes the production of antioxidant enzyme (CAT, SOD, GSH-Px) and reduced oxidative mediators (MDA, ROS), consistent with prior characterizations of perennial ryegrass rich in antioxidant compounds (Ali et al., 2024; Yang et al., 2019; Zulfiqar et al., 2025). Several Experimental studies used natural compounds (curcumin, rutin and dihydromyricetin), which enhanced the Nrf2 and exhibited inhibitory effects on NLRP3 mechanism (Bai et al., 2021; Bagherniya et al., 2021). These findings provide evidence that ryegrass effectively reduced UA-induced oxidative stress and inflammation, as key drivers of renal pathology (Liu et al., 2017; Lu et al., 2019; Wu et al., 2022).

Diets strongly influences the microbial composition, with higher microbial diversity generally indicating greater intestinal homeostasis and resistance to pathogenic invasion (Rehman et al., 2008; Wang et al., 2012). Gut microbiota analysis revealed that perennial ryegrass significantly enhanced microbial diversity and enriched multiple beneficial taxa, including Akkermansia, Shuttleworthia, Oscillibacter, Desulfovibrio, Lachnoclostridium, NK4A214 and butyricicoccus. These microorganisms are recognized for their roles in mucosal integrity, short-chain fatty acid (SCFA) production, anti-inflammatory signaling, and purine metabolism regulation. The increase in such taxa suggests improved intestinal barrier and metabolic stability, which may reduce systemic inflammatory load and contribute indirectly to renal protection (Ali et al., 2022; David et al., 2014; Guo et al., 2019). Network analysis demonstrating strong associations between these beneficial species and the PRD group described that ryegrass drives a functional restructuring of the microbial ecosystem. Given the growing recognition of the gut-kidney axis, these microbial changes likely play a significant role in moderating UA metabolism and renal inflammatory status.

Metabolomic profiling provided additional mechanistic insight into how ryegrass modulates host physiology. Ryegrass-fed goslings exhibited greater metabolic diversity and enrichment of pathways associated with amino acid metabolism, nucleotide metabolism, and ABC transporters pathways interconnected with antioxidant and immune regulation. Crucially, several bioactive antioxidant metabolites, including caffeic acid, kaempferol, skimmin, quercetin, ferulic acid, quinic acid, l-tryptophan, and l-proline, were significantly elevated in perennial ryegess-fed goslings. These compounds possess well-documented anti-inflammatory and radical-scavenging properties, suggesting that ryegrass enhances systemic antioxidant capacity not only through Nrf2 activation but also through increased availability of endogenous and microbially derived metabolites (Ali et al., 2024; Chilibroste et al., 2000; López-Andrés et al., 2014). Correlation analysis confirmed this functional relevance, as these antioxidant metabolites were positively associated with anti-inflammatory cytokines and antioxidant enzymes while inversely correlated with proinflammatory mediators and oxidative markers (Fu et al., 2024; Zulfiqar et al., 2025). These findings clearly demonstrate that ryegrass promotes a metabolite environment conducive to redox balance and immune modulation.

Conclusion

This study demonstrates that perennial ryegrass provides strong renoprotective effects in goslings by ameliorating high protein diet-induced HUA. Perennial ryegrass enhanced urate excretion through favorable regulation of renal transporters, reduced renal inflammation and fibrosis, and activated Nrf2-mediated antioxidant defenses while suppressing NLRP3 inflammasome signaling. It also improved gut microbial diversity and increased antioxidant metabolites that synergistically mitigated oxidative and inflammatory stress. Together, these integrated effects highlight the importance of diet-microbiota-host interactions and support perennial ryegrass as an effective, natural dietary strategy to prevent HUA-associated renal injury in geese production systems.

Ethics approval and consent to participate

Protocols used in animal collection were approved by the guidelines of the Henan Agriculture University Animal Care Committee.

Data availability

Data will be made available on demand.

CRediT authorship contribution statement

Muhammad Arslan Asif: Writing – original draft. Zeshan Zulfiqar: Writing – review & editing. Kai Jie Zhang: Visualization. Bahar E Mustafa: Formal analysis. Muhammad Ihtasham Asif: Data curation. Saira Saif: Methodology. Yalei Cui: Investigation. Liu Boshuai: Resources, Project administration. Yinghua Shi: Supervision, Resources, Project administration, Funding acquisition.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.