Recently, there has been a considerable increase in the global incidence of obesity due to modern lifestyle and changes in diet, such as consuming more saturated fat, lack of exercise, etc.(Reference Peng, Xie and Zhang1). Obesity has been identified as a principal driver in the development and progression of dyslipidaemia, cardiovascular and cerebrovascular diseases and type 2 diabetes(Reference Singh, Banerjee and Acosta2). The complications caused by obesity significantly affect the patient’s quality of life and may even be life-threatening. Furthermore, dyslipidaemia is one of the leading co-morbidities involving numerous primary and secondary renal diseases(Reference Mount and Juncos3). Lipid metabolism disorder results in lipid deposition in the kidney, which eventually progresses to chronic kidney injury. Emerging evidence suggests that lipid metabolism disorders, oxidative stress, inflammation and gut microbiota dysbiosis are key pathological mechanisms underlying chronic kidney injury.
A growing body of evidence suggests that chronic kidney injury is driven by lipid metabolism disorders, oxidative stress, inflammation and gut microbiota dysbiosis(Reference Yao, Li and Zeng4). Long-term consumption of a high-fat diet (HFD) elevates serum TAG and free fatty acid levels, promoting oxidative stress and inflammation, which are key pathological factors in renal injury(Reference Sun, Ge and Li5–Reference Chen, Wei and Li7). While the gut–kidney axis has been implicated in chronic kidney disease progression, the primary mechanisms of diet-induced renal injury involve direct metabolic disturbances(Reference Pongking, Haonon and Dangtakot8,Reference Hua, Han and Zhao9) .
Fucoxanthin (Fx), a marine xanthophyll-type carotenoid(Reference Terasaki, Kubota and Kojima10) (Fig. 1(a)), has demonstrated anti-inflammatory, antioxidant and anti-obesity properties(Reference Wu, Liou and Chen11–Reference Mao, Wang and Xiong13). Previous studies have shown that Fx protects against kidney injury by activating the Sirt1/Nrf2/HO-1 pathway(Reference Yang, Xing and Liu14) and restoring mitochondrial integrity(Reference Chen, Li and Hu15).
Effects of fucoxanthin (Fx) on body weight and kidney index in high-fat diet (HFD)-induced mice. (a) The chemical structure of Fx. (b) Schematic diagram of Fx treatment. (c) Mice’s body weight. (d) The Lee’s index of mice. (e) The kidney index of mice. Data are presented as mean ± standard deviation (sd) (n 12). Overall P-values for Lee’s index <0·001; Overall P-values for kidney index <0·002.
In this study, we hypothesise that Fx attenuates HFD-induced kidney injury by reducing oxidative stress and inflammation. An exploratory analysis of gut microbiota changes was additionally performed to provide preliminary data for future research. Our research aims to investigate the protective effects of Fx on renal function and lipid metabolism in an HFD-induced kidney injury mouse model.
Materials and methods
Reagents and fucoxanthin
Fucoxanthin was provided by Shandong Jiejing Group. Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), catalase (CAT), total cholesterol, TAG, nonesterified free fatty acid, uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), LDL-cholesterol, HDL-cholesterol assay kits and bicinchoninic acid protein assay kit were purchased from the Nanjing Jiancheng Bioengineering Institute. ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. Antibodies against ERK, p-ERK, p38, p-p38, JNK and p-JNK were obtained from Cell Signaling Technology Inc. Antibodies against NF-κB p65 and Caspase-1 were obtained from Proteintech Group, Inc., and NLRP3, ASC and cleaved-Caspase-1 were obtained from Affinity Biosciences Ltd (Affinity).
Animals and treatment
Forty-eight male C57BL/6J mice (16 ± 2 g, approximately 4 weeks old) were purchased from Zhejiang Provincial Experimental Animal Center (Certificate No.: SCXK 2014-0001). All experimental mice were raised in the SPF animal room under the following conditions: 24–26℃, 60 % humidity and 12 h light/dark cycle. The mice were fed freely with deionised water, and whole-nutrient sterilised feed was given daily for free eating. Experiments were conducted under the approval of the Animal Ethics Committee of Zhejiang Ocean University (Approval No. 2023168).
After 1 week of acclimatisation, twelve mice were randomly selected and freely fed an ordinary diet (control group), while the remaining mice were freely fed an HFD. The control group was fed a standard diet (D12451J, Research Diets Inc.) consisting of 19·2 % protein, 67·3 % carbohydrates and 4·3 % fat, with a fat-derived energy ratio of 10 %. The HFD (D12492, Research Diets Inc.) consisted of 26 % protein (casein), 35 % fat (24·5 % lard, 2·5 % soybean oil), 26 % carbohydrates (12·5 % maltodextrin, 6·9 % sucrose), with 5 % cellulose added, and a 60 % fat-derived energy ratio (online Supplementary Table S1). After 8 weeks, the mice with HFD were randomly divided into three groups (twelve mice in each group) to receive Fx gavage or normal saline once a day for 6 weeks as follows: (1) HFD + normal saline (HFD group), (2) HFD + 20 mg/kg Fx (Fx-20 group) and (3) HFD + 40 mg/kg Fx (Fx-40 group). The dosages of 20 mg/kg and 40 mg/kg were selected based on previous studies demonstrating the efficacy and safety of Fx in rodent models of metabolic disorders and organ injury. For a 60 kg human, this corresponds to a daily dose of approximately 97·3 mg. To minimise cage effects and avoid cross-contamination, mice from each treatment group were housed in separate cages. Multiple cages per group were used to control for cage-related bias, and samples from different cages were included in the microbiota analysis. The body weight of mice was recorded weekly. At the end of week 14, all mice were sacrificed after a 12-h fast.
The sample size for this study was determined based on pre-experimental data from our laboratory and similar studies in the literature. Previous research on HFD-induced kidney injury models typically used sample sizes of approximately twelve animals per group to detect significant biological effects. Our pre-experiment also indicated that twelve animals per group provided sufficient power to detect meaningful differences. In our pre-experiment, we defined ‘meaningful differences’ as a 20 % change in key renal function parameters (e.g. serum creatinine or blood urea nitrogen) compared with the control group, which is considered biologically significant in this model. Based on our pre-experimental data, we estimated an effect size (Cohen’s d) of 0·8 and an s d of 15 % for these parameters. Using G * Power software with a significance level (α) of 0·05 and a statistical power (1-β) of 80 %, we calculated that a sample size of twelve animals per group would be sufficient to detect these differences. Given the resources available and the experimental design, twelve animals per group were chosen to balance statistical power and feasibility, ensuring reliable data analysis while staying within the limits of available resources.
The kidney tissues of mice were rinsed with ice-cold saline immediately after dissection and then drained and weighed to calculate the kidney index. Blood and kidney tissues were collected and stored at –80℃ for later biochemical analysis. Faeces from mice were collected under sterile conditions for the 16S rRNA gene sequence.
$$\begin{gathered}{\text{Kidney index = (kidney weight/body weight) }} \times {\text{ 100% }} \hfill \\ {\text{Lee}}{\unicode{x2019}}{\text{s index = (}}\sqrt[3]{{{\text{body weight (g )}}}} \times {\text{ 100}}0)/{\text{body length (cm)}}{\text{.}} \hfill \\ \end{gathered}$$
Histopathological analysis
The kidney tissues of mice were harvested and fixed with 4 % paraformaldehyde. The tissue samples were dehydrated with alcohol, embedded in paraffin and cut into 4 μm sections, followed by haematoxylin–eosin staining, periodic acid–Schiff stain and Masson staining.
Serum biochemical analysis
Blood samples were collected from mice and centrifuged at 10 000 rpm for 5 min at 4°C to obtain sera. Serum levels of UA, CRE, BUN, TAG, total cholesterol, LDL-cholesterol, HDL-cholesterol, Lipopolysaccharide (LPS) and nonesterified free fatty acids were measured according to the manufacturer’s instructions.
Estimation of inflammatory cytokines and biochemical indices of oxidative stress
Kidney tissues of weight 200 mg were homogenised in ice-cold PBS to produce 10 % (w/v) kidney homogenates. The homogenates were then centrifuged (4°C) at 4000 rpm for 10 min to obtain supernatants. Protein concentrations in the supernatants were estimated using a bicinchoninic acid total protein assay kit. The contents of SOD, MDA, GSH-Px, T-AOC and CAT were determined following the manufacturer’s instructions. The levels of IL-1β and IL-6 were carried out using the ELISA kit according to the manufacturer’s protocol.
Western blotting analysis
Frozen kidney samples were thawed, chopped finely and precisely weighed (about 200 mg). The tissue samples were flash-frozen using liquid nitrogen and were then thoroughly ground in a mortar. The ground tissues were collected into sterile centrifuge tubes and mixed thoroughly with RIPA lysate containing protease inhibitors. Proteins from the kidney tissue lysates were collected and quantified using a bicinchoninic acid protein kit. Western blotting procedures were performed as previously reported19 using 50 μg protein samples. Proteins were visualised by ECL chemiluminescence (TransGen Biotech). Glyceraldehyde 3-phosphate dehydrogenase probed with the antibody was used as a control.
Immunohistochemical analysis
For immunohistochemistry, kidney slices were obtained as described in the previous section. The slices were deparaffinised with xylenes and hydrated and were incubated in 3 % hydrogen peroxide to eliminate endogenous enzymes. The tissue samples were boiled for antigen retrieval in an EDTA solution for 10 min. Using goat serum, kidney slices were blocked for 30 min at room temperature before being incubated with primary antibody NLRP3 (1:200 dilution, Cat No. DF7438, Affinity Biosciences), NF-κB p65 (1:150 dilution, Cat No. 80979-1-RR, Proteintech) overnight (4°C). Subsequently, sections were incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:500 dilution, Cat No. SA00001-2, Proteintech) for 30 min at 37°C, followed by signal amplification using a streptavidin–biotin complex kit (Cat No. SA1020, Boster Biological Technology). Diaminobenzidine (Cat No. ZLI-9018, ZSGB-BIO) was applied to visualise immunoreactive sites, and haematoxylin was used for counterstaining. Negative controls were included by omitting the primary antibody and replacing it with PBS. After washing with PBS, the sections were incubated with a horseradish peroxidase-conjugated secondary antibody (dilution 1:500) for 30 min at 25°C, followed by incubation with streptavidin–biotin complex working solution for 30 min. Immunoreactivity was visualised using diaminobenzidine, which produced brown staining at antigenic sites. Quantification of immunoreactivity was performed using ImageJ software (NIH). The mean optical density of positive staining was measured in five randomly selected fields per sample under 400× magnification. Results were expressed as relative intensity normalised to the control group.
Faecal microbiota analysis
Faecal samples were freshly collected from mice, immediately snap-frozen in liquid nitrogen and stored at –80°C until analysis. For 16S rRNA gene sequencing, four mice per group (n 4) were selected based on the following criteria: (1) body weight closest to group average, (2) no signs of diarrhoea or abnormal behaviour and (3) from different cages to minimise cage effect bias. This ensured the representativeness and comparability of the faecal samples for microbiota analysis.
Microbial DNA was extracted using the Cetyltrimethylammonium bromide/Sodium dodecyl sulfate (CTAB/SDS) method, and DNA purity and concentration were assessed by agarose gel electrophoresis and spectrophotometry. The V3–V4 regions of the bacterial 16S rRNA gene were amplified using barcoded universal primers (338F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). PCR products were purified and sequenced using the Illumina MiSeq platform with paired-end 250 bp reads. Raw sequences were demultiplexed, quality filtered and merged using FLASH. After removal of chimeric sequences, high-quality reads were clustered into operational taxonomic units (OTU) with 97 % similarity using Uparse (v7·0·1001). The representative sequences of each OTU were annotated against the SILVA database (v138) using the Mothur method. Alpha diversity metrics (Chao1, Shannon, Simpson) and beta diversity (Bray-Curtis distance, non-metric multidimensional scaling and PCoA) were computed using QIIME2 (v2022·8). One-way ANOVA or Kruskal–Wallis test was used for group comparisons, followed by post hoc Dunn’s test when applicable. False discovery rate correction was applied for multiple testing. Data visualisation was done using R (v4·2·1) with the ‘vegan’ and ‘ggplot2’ packages.
Statistical analysis
The data are presented as mean ± standard deviation (s d). Statistical analyses were performed using IBM SPSS Statistics 26·0. Prior to one-way ANOVA, data were tested for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test. If both assumptions were met, one-way ANOVA followed by Tukey’s post hoc test was used to compare differences among groups. In cases where assumptions were not satisfied, non-parametric tests (e.g. Kruskal–Wallis with Dunn’s post hoc test) were applied as appropriate. P < 0·05 indicates significant differences, and P < 0·01 indicates extremely significant differences.
Results
Fucoxanthin decreases body weight, kidney index and Lee’s index in mice
The experiment was performed over a period of 14 weeks, with the grouping of experimental animals shown in Fig. 1(b). In the initial phase, the average weight of the mice was 17·58 ± 1·19 g. After 8 weeks of the HFD diet, the average weight of the HFD group increased significantly to 28·28 ± 0·88 g (v. the control group 22·85 ± 1·01 g). At the end of the treatment, the weight of mice in the Fx treatment group decreased significantly to 25·80 ± 1·51 g (Fx 20 mg/kg) and 25·64 ± 1·56 g (Fx 40 mg/kg) (Fig. 1(c)). Furthermore, Lee’s index and kidney index were also significantly increased in the HFD group, which was found to be reversed in Fx treatment (Fig. 1(c)–(d)).
Fucoxanthin improves kidney function and dyslipidaemia in mice
We measured the serum levels of UA, CRE and BUN in mice to evaluate the ameliorating effect of Fx on HFD-induced renal injury. Mice in the HFD group showed significantly increased serum levels of UA, CRE and BUN (Fig. 2(a)–(c)), indicating that long-term HFD results in renal insufficiency. Fx treatment significantly reduced the above indices. Concomitantly, Fx treatment reduced the levels of total cholesterol, TAG, nonesterified free fatty acid and LDL-cholesterol and increased the level of HDL-cholesterol when compared with the HFD group (Fig. 2(d)–(h)). These observations suggest that Fx can effectively improve HFD-induced renal injury and regulate dyslipidaemia in mice.
Fucoxanthin (Fx) improves renal dysfunction and dyslipidaemia in high-fat diet (HFD)-induced mice. Serum uric acid (UA) (a), creatinine (CRE) (b), blood urea nitrogen (BUN) (c), total cholesterol (TC) (d), TAG (e), nonesterified free fatty acid (NEFA) (f), LDL-cholesterol (g) and HDL-cholesterol (h). Data are presented as mean ± standard deviation (sd) (n 12). Overall P-values for UA, CRE, BUN, TC, TAG, NEFA, LDL-cholesterol and HDL-cholesterol < 0·001.
Fucoxanthin ameliorates renal structure in high-fat diet-induced kidney injury mice
Histopathological analyses of mouse kidneys were performed to assess the effect of Fx on HFD-induced pathological injury. The renal bodies in the control group consisted of round and spherical renal vesicles surrounding the glomeruli with clear lumens. The renal tubules had regular morphology without any inflammatory infiltration. In contrast, the HFD group displayed glomerular atrophy and enlargement of cystic cavities. The structural clarity of the tubules was compromised as evidenced by renal tubular vacuolisation and conspicuous inflammatory cell infiltration in the surrounding regions. In the Fx-20 group, proliferation of mesangial cells and increases in mesangial matrix were inhibited, and the vacuolisation of renal tubules was improved. In the Fx-40 group, the morphology and structure of the glomerulus and tubules were clear without any inflammatory cell infiltration (Fig. 3(a)). Periodic acid–Schiff staining of the mesangial membrane in the glomeruli of the HFD group was significantly increased, the mesangial area was significantly enlarged and the basement membrane around the renal tubules was significantly thickened. After Fx treatment, the periodic acid–Schiff positive sites in the glomeruli of mice were significantly reduced, and basement membrane hyperplasia around the renal tubules was improved (Fig. 3(b)). Collagen deposition in renal tissue was detected by Masson staining. As shown in Fig. 3(c), a large amount of collagen was deposited in the kidney tissue of mice in the HFD group along with renal tubulointerstitial fibrosis. After Fx administration, no substantial interstitial fibrosis was detected. These results suggested that Fx treatment ameliorates HFD-induced renal structural changes and alleviates renal damage in mice.
Fucoxanthin (Fx) ameliorates renal structure in high-fat diet (HFD)-induced kidney injury mice. (a) Haematoxylin–eosin (HE) staining images of the kidney. (b) Periodic acid–Schiff (PAS) staining images of the kidney. (c) Masson staining images of the kidney.
Fucoxanthin attenuates renal oxidative stress and inflammation in high-fat diet-fed mice
Mice administered HFD for 8 weeks showed decreased SOD, GSH-Px and T-AOC, CAT levels and increased MDA levels and serum LPS levels. In agreement with these, the levels of IL-1β and IL-6 in the HFD group were significantly increased. These observations indicate that long-term HFD can lead to severe lipid peroxidation and inflammatory response in the kidney. Importantly, Fx treatment dose-dependently reduced the elevated serum LPS levels (Fig. 4(h)), suggesting its protective effect on gut barrier integrity. In comparison, the levels of MDA and the two proinflammatory cytokines were significantly reduced in all Fx groups (Fig. 4(e)–(g)), with the higher dose (40 mg/kg) showing more pronounced effects. Moreover, Fx also increased the levels of SOD, GSH-Px, T-AOC and CAT in the kidney to almost normal levels (Fig. 4(a)–(d)).
Fucoxanthin (Fx) attenuates renal oxidative stress and inflammatory cytokine levels in high-fat diet (HFD)-fed mice. Kidney tissue superoxide dismutase (SOD) (a), glutathione peroxidase (GSH-Px) (b), T-AOC (c), catalase (CAT) (d), malondialdehyde (MDA) (e), IL-6 (f), IL-1β (g) and serum LPS (h). Data are presented as mean ± standard deviation (sd) (n 12). V Overall P-values for SOD, GSH-Px, T-AOC, CAT, MDA, IL-6, IL-1β and LPS < 0·001.
Effects of fucoxanthin on NLRP3 inflammasome in the kidney
In this study, the protein expression of the NLRP3 inflammasome was also analysed. When compared with the control group, the protein levels of NLRP3, ASC and cleaved-Caspase-1 in the HFD group were significantly increased. Fx treatment was found to reduce the expression of the above proteins (Fig. 5(b)–(g)). Similar results were obtained by immunohistochemical identification of NLRP3 expression in kidney sections, which is mainly expressed in renal tubules (Fig. 5(a) and (g)).
Effects of Fx on NLRP3 inflammasome in the kidney. (a) Immunohistochemistry staining of NLRP3 in the kidney tissues, 400 × magnification. (b) Western blot analysis of NLRP3 inflammasome. Densitometric analysis of NLRP3 (c), ASC (d), Caspase-1 (e) and cleaved-Caspase-1 (f). (g) Quantitative analysis of NLRP3 immunohistochemical staining. Data are presented as mean ± standard deviation (sd) (n 12). Overall P-values for NLPR3, ASC, Caspase-1, Cleaver-Caspase-1 and NLRP3 < 0·001. HFD, high-fat diet; Fx, fucoxanthin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Effects of fucoxanthin on MAPK pathway in the kidney
We also performed western blotting analysis of MAPK pathway-related proteins. After long-term HFD feeding, the relative protein levels of p-ERK, p-JNK and p-p38, as well as NF-κB p65, were significantly up-regulated in the HFD group (Fig. 6(b)–(g)). Fx treatment significantly reversed these changes. Concomitantly, immunohistochemical staining showed that treatment with Fx significantly reduced the HFD-induced NF-κB p65 positive sites in the kidney tissue (Fig. 6(a) and (g)).
Effects of Fx on the MAPK pathway in the kidney. (a) Immunohistochemistry staining of NF-κB p65 in the kidney tissues, magnification: ×400. (b) Western blot analysis of MAPK pathway-related proteins. Densitometric analysis of p-ERK/ERK (c), p-p38/p38 (d), P-JNK/JNK (e) and NF-κB p65 (f). (g) Quantitative analysis of NF-κB p65 immunohistochemical staining. Data are presented as mean ± standard deviation (sd) (n 12). Overall P-values for p-ERK/ERK, p-p38/p38, P-JNK/JNK and NF-κB p65 and quantitative analysis of NF-κB p65 immunohistochemical staining < 0·001. HFD, high-fat diet; Fx, fucoxanthin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fucoxanthin regulates the composition of intestinal microbes
We investigated whether Fx improvement of HFD-induced kidney injury was associated with a regulatory effect on the composition of the intestinal microbes. As demonstrated in Fig. 7(a), the data volume of sequenced reads was reasonable due to a flat rarefaction curve. 16S rRNA sequencing analysis revealed a total of 844 OTU, among which each group accounted for 89·8, 55·94, 83·88 and 79·15 % of the total OTU. Venn diagram analysis showed that the number of OTU co-occupied by the HFD group and control group was lower (accounting for 49·17 %), indicating that the microbial composition of these two groups was significantly different. However, the OTU of the Fx-20 group and the Fx-40 group had a high degree of overlap with the control group at 78·79 and 75·00 %, respectively, indicating that the microbial composition of these three groups was highly similar (Fig. 7(b)).
Fx regulates the composition of intestinal microbes. (a) Rarefaction curve. (b) Venn diagrams of the sample. (c) The Chao index. (d) The Ace index. (e) The Shannon index. (f) The Simpson index. (g) Non-metric multidimensional scaling analysis (NMDS) analysis. (h) Hierarchical cluster analysis. Data are presented as mean ± standard deviation (sd) (n 12). Overall P-values for the Chao index, Shannon index and Ace index < 0·001. Overall P-values for the Simpson index < 0·029. OTU, operational taxonomic unit; HFD, high-fat diet; Fx, fucoxanthin.
Furthermore, we also calculated the α-diversity index in this study. In the HFD group, the Chao, Ace and Shannon indices were significantly decreased, while the Simpson index was significantly increased, indicating that long-term HFD feeding resulted in a decrease in intestinal microbial diversity and richness. As expected, Fx reversed this negative effect (Fig. 7(c)–(f)). The beta diversity of each group was analysed by non-metric multidimensional scaling analysis and hierarchical clustering analysis. In the non-metric multidimensional scaling plot, the HFD and control groups were considerably distant, with no areas of crossover suggesting a complete divergence in microbial composition between the two groups. The trajectory of the Fx group displayed a gradual shift towards the normal group, even demonstrating an overlap, thus indicating that Fx can rectify the beta diversity of gut microbiota (Fig. 7(g)). As shown in Fig. 7(h), the four groups of gut microbiotas can be divided into two main groups: Group 1 (HFD) and Group 2 (Control, Fx-20 and Fx-40), consistent with non-metric multidimensional scaling analysis. This indicates that the microbial composition of the HFD group diverges from the control group and that after Fx treatment, the microbial composition gravitates towards that of the control group. Fx ameliorated HFD-induced gut microbiota dysbiosis in mice at the phylum and family levels.
Discussion
Obesity and the duration of obesity are associated with increased risks of numerous chronic diseases such as CVD, cancer, diabetes and chronic kidney disease(Reference Sterling and Bowen16,Reference Ananthakrishnan and Kaysen17) . In fact, recent studies have suggested a strong relationship between obesity and decreased renal function(Reference Yang, Li and Peng18). Fx has been reported to improve diabetic renal function and renal fibrosis by reducing oxidative stress(Reference Li, Chen and Jiao19). Compared with other well-known antioxidants, such as vitamin C, vitamin E and N-acetylcysteine, Fx exhibits unique advantages. For instance, while vitamin C and vitamin E primarily function by directly scavenging free radicals, Fx not only possesses direct antioxidant capacity but also enhances the endogenous antioxidant defense system by activating the Nrf2/ARE pathway(Reference Yang, Li and Yan20). Specifically, Fx contains an allenic bond and conjugated carbonyl groups in its molecular structure, which not only endow it with direct antioxidant activity but also enable it to activate the Nrf2/ARE signalling pathway more potently than other carotenoids(Reference Mao, Wang and Xiong13). In addition, several comparative studies have reported that Fx has a higher renal accumulation and longer biological half-life than β-carotene or lutein(Reference Yang, Li and Peng18). These structural and pharmacokinetic advantages may contribute to Fx’s greater capacity to modulate oxidative stress and inflammatory responses in kidney tissues.
In the present study, Fx mitigated HFD-induced kidney injury through a multifaceted mechanism, including antioxidative effects, improved lipid metabolism and suppression of inflammatory pathways. Key markers of renal dysfunction, such as elevated BUN, CRE, UA and increased kidney index, were significantly improved by Fx treatment, suggesting effective nephroprotection. Elevated levels of these markers indicate impaired renal function.
HFD-induced excess free fatty acids are converted to TAG, which can damage cells and recruit inflammatory cells, creating an inflammatory microenvironment(Reference Luo, Chen and Liu21). Luo et al. found that ferrostatin-1 (ferroptosis inhibitor), a ferroptosis inhibitor, alleviates HFD-induced liver and kidney injury by reducing pathological damage, inflammatory cell infiltration and fibrosis(Reference Wang, Liu and Zhang22). Similarly, our study showed that Fx treatment suppressed HFD-induced increases in body weight and kidney index while improving renal function, dyslipidaemia and renal structure.
HFD accelerates radical formation in vivo, disrupting intrinsic antioxidant defences and leading to systemic oxidative stress and a proinflammatory state(Reference Song, Liu and Zhang23). Free radicals and inflammation are key contributors to kidney injury under long-term HFD conditions. Natural antioxidant and anti-inflammatory agents, such as Fx, are promising interventions. Malondialdehyde (MDA), a lipid peroxidation product, reflects oxidative stress levels(Reference Song, Liu and Zhang23). Antioxidant enzymes, including SOD, GSH-Px and CAT, protect against oxidative damage(Reference Yamamoto, Takabatake and Takahashi24). In this study, Fx effectively reduced the MDA content and restored the activities of SOD, GSH-Px, T-AOC and CAT in the kidney of HFD-fed mice.
NLRP3 inflammasome is a complex of multiple proteins, including NLRP3, ASC and pro-Caspase-1(Reference Zhang, Tao and Xia25), which activates Caspase-1 to mediate the maturation and secretion of proinflammatory cytokines(Reference Komada and Muruve26). Its activation is implicated in acute kidney injury, calcium oxalate nephropathy and chronic kidney disease. Inhibition of NLRP3 inflammasome activation and reactive oxygen species production can alleviate kidney injury. In our study, HFD triggered NLRP3 inflammasome activation in renal tissue, while Fx significantly suppressed the expression of NLRP3, ASC, Caspase-1 and cleaved-Caspase-1. Additionally, Fx reduced pro-fibrotic cytokines such as TNF-α, IL-6 and IL-1β, suggesting its role in alleviating oxidative stress and inflammation.
The MAPK pathway, including p38 MAPK, ERK and JNK subfamilies, regulates renal injury(Reference Rajamaki, Mayranpaa and Risco27). NLRP3-mediated(Reference Ma, Xu and Zhu28) IL-1β secretion activates p38 MAPK(Reference Wang, Zhang and Yu29), which enhances renal injury via the NF-κB pathway(Reference Hedl and Abraham30). ERK activation can also exacerbate kidney injury by inducing hemodynamic changes(Reference Foley, Zlitni and Denou31). Our findings show that Fx attenuated the phosphorylation of p38 MAPK, JNK and ERK in HFD-fed mice, indicating that MAPK pathway inhibition is a key mechanism of Fx-mediated renal protection.
To preliminarily explore the role of gut microbiota in the gut–kidney axis under HFD conditions, we conducted exploratory 16S rRNA sequencing on a subset of animals (n 4/group). Although limited by a small sample size and not designed for definitive conclusions, this analysis provides initial insights into microbial shifts following HFD and Fx treatment.
Nonetheless, several limitations of our study must be acknowledged. First, the HFD model used may not fully mimic human dietary patterns, which may limit translational relevance. Second, while serum creatinine and BUN are established biomarkers in murine models, they do not fully align with clinical markers such as albuminuria or creatinine clearance. Third, as mentioned, the gut microbiota analysis was conducted on a limited number of faecal samples and should be considered exploratory, not conclusive. Additionally, Fx-treated groups exhibited reduced food intake, which may have influenced weight loss and renal protection independent of Fx itself. Pair-feeding experiments or caloric-matched controls are warranted in future studies to control for this potential confounder. Finally, a more detailed investigation into microbial metabolites and their effects on renal health is needed to elucidate potential mechanistic links between the gut and kidney.
Conclusions
In summary, our study demonstrates that Fx alleviates HFD-induced kidney injury in mice by mitigating oxidative stress, reducing inflammation and modulating the MAPK and NLRP3 signalling pathways. These findings highlight Fx’s potential as a nutraceutical agent for protecting against obesity-related renal dysfunction. Although preliminary microbial profiling suggests that Fx may also influence the gut–kidney axis, the gut microbiota findings are exploratory and require further validation in larger cohorts and mechanistic studies. Overall, this work provides a foundation for future investigations into the multifaceted protective effects of Fx and its translational potential in the prevention of metabolic kidney disease.
Supplementary material
For supplementary material/s referred to in this article, please visit https://doi.org/10.1017/S0007114525105023.
Acknowledgements
We thank the following funders for their support of this work:
Zhoushan Municipal Public Welfare Technology Projects (grant no. 2023C31021) and Zhejiang Provincial Medical and Health Technology Plan Project (grant no. 2023RC109).
Z. L. and B. X. contributed equally to this work and share first authorship. H. S., Z. L. and Z. Y. designed the study. B. X., J. Z. and Y. T. implemented and performed the analysis. H. S. and B. X. wrote the manuscript. B. X. and J. Z. prepared the tables and figures. All authors discussed the results. All authors contributed to and revised the manuscript critically.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
