Kidney tea [Orthosiphon aristatus (Blume) Miq.] improves diabetic nephropathy via regulating gut microbiota and ferroptosis

Introduction Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Due to its complex pathogenesis, new therapeutic agents are urgently needed. Orthosiphon aristatus (Blume) Miq., commonly known as kidney tea, is widely used in DN treatment in China. However, the mechanisms have not been fully elucidated. Methods We used db/db mice as the DN model and evaluated the efficacy of kidney tea in DN treatment by measuring fasting blood glucose (FBG), serum inflammatory cytokines, renal injury indicators and histopathological changes. Furthermore, 16S rDNA gene sequencing, untargeted serum metabolomics, electron microscope, ELISA, qRT-PCR, and Western blotting were performed to explore the mechanisms by which kidney tea exerted therapeutic effects. Results Twelve polyphenols were identified from kidney tea, and its extract ameliorated FBG, inflammation and renal injury in DN mice. Moreover, kidney tea reshaped the gut microbiota, reduced the abundance of Muribaculaceae, Lachnoclostridium, Prevotellaceae_UCG-001, Corynebacterium and Akkermansia, and enriched the abundance of Alloprevotella, Blautia and Lachnospiraceae_NK4A136_group. Kidney tea altered the levels of serum metabolites in pathways such as ferroptosis, arginine biosynthesis and mTOR signaling pathway. Importantly, kidney tea improved mitochondrial damage, increased SOD activity, and decreased the levels of MDA and 4-HNE in the renal tissues of DN mice. Meanwhile, this functional tea upregulated GPX4 and FTH1 expression and downregulated ACSL4 and NCOA4 expression, indicating that it could inhibit ferroptosis in the kidneys. Conclusion Our findings imply that kidney tea can attenuate DN development by modulating gut microbiota and ferroptosis, which presents a novel scientific rationale for the clinical application of kidney tea.


Introduction
Diabetes and its various complications seriously threaten the health of patients and impose a huge medical and economic burden on society.According to statistics, there were approximately 537 million adults with diabetes globally in 2021, and this number will rise to 783 million by 2045 (Sun et al., 2022).Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes, accounting for 40% of diabetic patients (Smyth et al., 2022).It is mainly characterized by hypertension, progressive proteinuria, and decreased renal function, ultimately developing into end-stage renal disease.Increasing evidence suggests that multiple pathological mechanisms, such as oxidative stress (Liu et al., 2023), chronic inflammation (Qiu et al., 2023), mitochondrial dysfunction (Zhou et al., 2023) and excessive ferroptosis (Li et al., 2023), play crucial roles in the occurrence and progression of DN.Currently, the therapeutic strategies that limit the development of DN include anti-hyperglycemic sodiumglucose cotransporter 2 (SGLT2) inhibitors, as well as antihypertensive angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) (Zhang et al., 2022a;Barrera-Chimal et al., 2022;Vergara et al., 2023).Due to the complex pathological mechanisms, it is urgent to develop effective agents for DN management.
Orthosiphon aristatus (Blume) Miq., also known as kidney tea, is a perennial herb belonging to the family Lamiaceae (Zhu et al., 2023).Kidney tea is recorded in the traditional Dai medical works "Bei Ye Jing" and "Dang Haya," and has been used for more than 2,000 years.It is traditionally consumed as a functional tea in southwestern China and Southeast Asia, owing to its distinctive aromatic odour (Ashraf et al., 2018).On the other hand, this timehonoured medicine is widely used to treat kidney diseases, gout and diabetes (Hsu et al., 2010;Ching et al., 2013;Chen et al., 2020).In a previous study, we identified five compounds from kidney tea and confirmed that rosmarinic acid might play a hypoglycemic effect by regulating the activity of α-glucosidase (Zhu et al., 2021).Another research found that aqueous and methanolic extracts of kidney tea could reduce blood glucose levels in diabetic mice via increasing glucose transporter 4 (GLUT4) translocation to the plasma membrane (Bassalat et al., 2023).Furthermore, the phenolic acids might be the active ingredients of kidney tea that exerted antidiabetic effects, and the mechanisms were related to antioxidant activity, inhibition of α-glucosidase and α-amylase, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-mediated glucose uptake (Wang et al., 2023).However, the mechanisms by which kidney tea improves DN have not been elucidated.
Gut microbiota is the microorganisms in the human gastrointestinal tract, which is vital for maintaining the health of the host.A large number of studies have found that the gut microbiota is involved in the pathogenesis of DN (Zhang et al., 2022b;Cai et al., 2022;Zhao et al., 2023).DN patients exhibited dysbiosis in the gut microbiota, manifested by increased levels of Lactobacillus, Megaphaera and Sutterella, as well as decreased levels of Lachnochlostridium and Roseburia (Du et al., 2021).Recent research has demonstrated that gut microbiota-derived outer membrane vesicles can promote DN progression by activating tubulointerstitial inflammation (Chen et al., 2023).Notably, the diversity of the gut viruses was significantly reduced in DN patients (Fan et al., 2023).In addition, non-targeted metabolomics revealed that plasma kynurenine, gluconolactone, CE-C0218 and CE-A0242, as well as urinary threonic acid, sphingomyelin, 1-methylpyridin-1ium and 1-palmitoyl-glycero-3-phosphocholine could be potential biomarkers for DN (Hirakawa et al., 2022).These findings imply that the changes in the composition of gut microbiota and metabolites are involved in the pathogenesis of DN, and that interventions targeting them may provide new therapeutic strategies for DN.Here, we hypothesized that kidney tea had the potential to regulate gut microbiota and host metabolic function, thereby exerting a therapeutic effect on DN.Therefore, we investigated the protective effects of kidney tea on DN mice and elucidated the possible mechanisms by combining gut microbiota and serum metabolomics analysis.

Sample preparation
Kidney tea was collected from Yunnan Province, China, and then identified by Prof. Binghong Fei.Kidney tea (600 g) was soaked in 6,000 mL of water for 1 h, followed by decoction for 30 min and filtration.This process was repeated twice, and the combined extract was concentrated to 1 g/mL using a rotary evaporator and then stored in a refrigerator at −20 °C for subsequent experiments.
Thermo Q-Exactive HFX (Thermo Fisher Scientific) was used for MS analysis, and the electrospray ionization (ESI) source was used for detection in positive and negative ion modes.The parameters of ESI source were set as follows: the spray voltage was 3.8 kV (+) and 3.0 kV (−), the source temperature was 320 °C, and the range of MS scan was 90-1,300 m/z.

Animals and drug intervention
The animal experiment was approved by the Life Science Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. 2024-KY-0015-002).30 male C57BLKS/J db/db and 6 male C57BLKS/J db/m mice (7-week-old) were obtained from Nanjing Junke Bioengineering Co., Ltd.(Nanjing, China).db/db mouse exhibits leptin receptor deficiency and often serves as a common animal model for spontaneous type 2 diabetes (Suriano et al., 2021).All mice were maintained in an SPF-grade environment with a temperature of 22 °C ± 2 °C, a relative humidity of 50% ± 5%, and a 12 h light/dark cycle.All animals had free access to standard feed and water with 1 week of acclimatization.Subsequently, 30 db/db mice were randomly allocated into five groups (n = 6), namely, the model group (Mod), the kidney tea low-dose group (KTL), the kidney tea medium-dose group (KTM), the kidney tea high-dose group (KTH), and the dapagliflozin group (DAPA).Moreover, 6 db/ m mice were used as the control group (Con).Of these, the KTL, KTM and KTH groups were given 2, 4 and 8 g•kg −1 •d −1 kidney tea extract, respectively.The DAPA group was given 1.3 mg•kg −1 •d −1 dapagliflozin.After 1 week of adaptive feeding, the mice in each intervention group received the corresponding drug, and the mice in the Con and Mod groups received distilled water.All mice were gavaged with a volume of 10 mL•kg −1 and administered once daily for 8 weeks.

Sample collection
The blood from the tail vein was collected after an 8-hour fast at 0, 2, 4, 6, and 8 weeks, and the fasting blood glucose (FBG) levels were measured by a glucometer (Bayer Healthcare LLC, NY, United States).The urine was collected at week 8 by metabolic cages.Ultimately, the mice were anaesthetized with pentobarbital sodium, the eyeballs were removed for blood collection, and serum was obtained by centrifugation.After the mice were executed, the kidneys and contents from the ileocecal junction were collected.

Histopathologic examination
The kidney tissues of appropriate size were fixed in 4% paraformaldehyde, and the fixed tissues were embedded in paraffin.The samples were sectioned and stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and Masson, and subsequently observed under light microscope for renal histopathological changes.
The FASTQ format data were processed through QIIME 2 pipeline.Taxonomic assignment of amplicon sequence variants (ASVs) was performed based on the SILVA v138 16S database.The composition of species in the samples was analyzed based on the results of species annotation.Principal coordinates analysis (PCoA) was used to assess the beta diversity, and linear discriminant analysis effect size (LEfSe) analysis was performed to further screen significantly different species within different groups.
Orthogonal partial least squares discriminant analysis (OPLS-DA) and principal component analysis (PCA) were performed to evaluate the overall distribution trend among groups.Variable importance in the projection (VIP) value > 1 and p < 0.05 (Student's t-test) were utilized to identify significantly differential metabolites, and Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for pathway enrichment analysis.

Electron microscopy
A 1 mm 3 volume of renal tissue was fixed overnight in 2.5% glutaraldehyde.After dehydration, embedding, curing and sectioning, ultrathin sections were stained with 2% uranyl acetate and lead citrate.Subsequently, cellular ultrastructural changes were observed under a transmission electron microscope (Hitachi HT7700, Tokyo, Japan).

Determination of MDA, 4-HNE and SOD in kidney tissues
The kidney tissues were homogenized in PBS and used for the determination of ferroptosis-related markers.The activity of superoxide dismutase (SOD) was measured by SOD activity assay kits (Elabscience Biotechnology Co., Ltd., Wuhan, China), and the levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) were measured via MDA and 4-HNE assay kits purchased from Nanjing Jiancheng (Nanjing, China), respectively.

Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR)
0Kidney tissues were washed with PBS, and TRIzol reagent was used to extract the total RNA.The above RNA was reverse transcribed by Supermo III M-MLV reverse transcriptase to obtain the corresponding cDNA.PCR amplification was carried out using 2 × Power Taq PCR MasterMix and corresponding primers.The amount of PCR products was calculated using the fluorescence intensity of SYBR Green I, and the results were processed using the 2 −ΔΔCT method.These reagents were purchased from Beijing BioTeke Biotechnology Co., Ltd.(Beijing, China).Primer sequences were shown in Supplementary Table S1.

Western blotting analysis
Kidney tissues were added to RIPA buffer containing 1% PMSF, which were incubated at 4 °C for 30 min.The proteins were obtained by centrifugation at low temperature.After separation, the protein samples were transferred to polyvinylidene difluoride membranes.Subsequently, the membranes were sealed with 5% skimmed milk and incubated with primary antibody overnight.After washing with TBST buffer solution, the membranes were incubated with secondary antibody.The primary antibodies were as follows: anti-GPX4 (Affinity Biosciences, Changzhou, China), anti-ACSL4 (Proteintech, Wuhan, China), anti-NCOA4 (Santa Cruz, Dallas, Texas, United States), anti-FTH1 (BOSTER Biological Technology Co., Ltd., Wuhan, China) and anti-β-actin (Proteintech, Wuhan, China).

Statistical analysis
GraphPad Prism 9.0 was used for statistical analysis.Data were presented as mean ± SD.One-way ANOVA was used to analyze the significance, followed by Dunnett's test.p < 0.05 was considered statistical significant.Moreover, the potential relationships between gut microbiota, serum metabolites and host phenotypes were performed via Spearman correlation analysis.

Chemical composition of kidney tea
Based peak intensity (BPI) chromatograms were shown in Supplementary Figure S1.Polyphenols are the main active ingredients of kidney tea (Chiang et al., 2023), and such compounds are considered to have antioxidant and nephroprotective effects (Jiang et al., 2012;Oršolić et al., 2021).Therefore, we identified the polyphenols in kidney tea.12 polyphenols with relative percentages higher than 0.5% were identified by qualitative analysis (Table 1).Among them, the relative percentage of rosmarinic acid was the highest, accounting for 6.40%.The total content of 12 identified polyphenols in kidney tea reached 19.76%.

Therapeutic effects of kidney tea on DN mice
The animal experimental design was described in Supplementary Figure S2.First, we evaluated the therapeutic effects of kidney tea on DN mice via measuring FBG.Compared to normal mice, DN mice exhibited significant hyperglycemia, whereas kidney tea treatment reduced FBG levels to some extent (Figure 1A).Chronic inflammation is closely related to the pathogenesis of DN (Zhang et al., 2024).Therefore, the levels of the inflammatory factors IL-6, IL-10 and TNF-α were measured.The results found that TNF-α and IL-6 levels were significantly increased, while anti-inflammatory cytokine IL-10 levels were significantly decreased in DN mice (Figures 1B-D).High and medium doses of kidney tea could obviously reverse the levels of these cytokines.Moreover, renal injury-related markers including Scr, BUN and urinary albumin were measured.Ferroptosis has been reported to occur in renal tubular epithelial cells in diabetic nephropathy (Jin and Chen, 2022).Therefore, we also measured NGAL and NAG levels.The levels of Scr, BUN, urinary albumin, NGAL and NAG in the Mod group were significantly higher than those in the Con group, which could be significantly reduced by kidney tea treatment (Figures 1E-I).
Next, the effects of kidney tea on renal morphological changes in DN mice were determined by H&E, PAS and Masson staining (Figures 2A-C).By H&E staining, shrunken glomeruli, interstitial inflammatory infiltrate, and massive degeneration of tubular epithelium were observed in DN mice (Figure 2A).PAS and Masson staining showed thickened glomerular basement membrane and increased collagen fiber proteins in DN mice, respectively (Figures 2B,C).Notably, these pathological changes were ameliorated after kidney tea treatment.Taken together, these results suggested that kidney tea improved renal function and injury in DN mice.

Kidney tea modulated the gut microbiota in DN mice
Considering that DN mice treated with high-dose kidney tea showed the best renal protection, the KTH group was used for subsequent gut microbiota and metabolomics analysis.We first investigated the composition of the gut microbiota after kidney tea treatment by 16S rDNA gene sequencing.PCoA analysis found an obvious separation between the clusters of the Con, Mod and KTH groups (Figure 3A), indicating that kidney tea caused significant changes in the composition of the gut microbiota.At the phylum level, Bacteroidota, Desulfobacterota, Firmicutes, and Actinobacteriota were the dominant species among all of the groups (Figure 3B).Subsequently, we examined the structural alterations at the genus level (Figure 3C).The results revealed that the relative abundance of Muribaculaceae, Lachnoclostridium, Akkermansia, Corynebacterium and Prevotellaceae_UCG-001 was increased, while the relative abundance of Blautia, Alloprevotella and Lachnospiraceae_NK4A136_group was decreased in DN mice compared to normal mice (Figures 3D-K).Significantly, kidney tea administration could reverse the dysbiosis of these bacterial taxa to some extent.Furthermore, LEfSe analysis with an LDA score ≥ 3 was used to identify the characteristic bacterial taxa of each group (Figure 3L).Our results indicated that the significantly different microbiota in the Con, Mod and KTH groups were 9, 15 and 36, respectively.Of these, f_Muribaculaceae, c_Verrucomicrobiae and g_Akkermansia were the dominant microbiota in the Mod group.Importantly, highdose kidney tea could enrich the relative abundance of o_ Bacteroidales, g_Bacteroides, f_Rikenellaceae, g_Rikenellaceae_ RC9_gut_group, and f_Tannerellaceae.

Kidney tea regulated serum metabolite profiles in DN mice
In addition, we used untargeted serum metabolomics to investigate the effects of kidney tea on host metabolites.The PCA plots showed the distinct separation of each group under positive and negative ion modes (Figures 4A, B).Meanwhile, VIP value > 1 and p < 0.05 were used as screening criteria for significantly differential metabolites.A total of 105 differential metabolites were identified in the Mod group compared with the Con group, indicating that the metabolite profile changed significantly during the development of DN.Compared with the Mod group, kidney tea intervention downregulated 41 metabolites and upregulated 31 metabolites.The heatmap showed the 50 most differentiated serum metabolites, implying that kidney tea exerted a profound effect on host metabolism (Figure 4C).Therefore, we further investigated the function of the differential metabolites between the two groups with pathway enrichment analysis.The results found that kidney tea mainly participated in the regulation of metabolic pathways such as ferroptosis, arginine biosynthesis, and mammalian target of rapamycin (mTOR) signaling pathway (Figure 4D).Among them, 14 differential metabolites were from the top 11 metabolic pathways (Supplementary Table S2).Notably, the relative abundance of serotonin, deoxyinosine, saccharin and 1-palmitoyl-2-oleoyl-snglycerol was increased, while the relative abundance of isopentenyl pyrophosphate and N-alpha-acetyl-l-ornithine was decreased in DN mice compared with normal mice (Figures 4E-J).Kidney tea intervention reversed the alterations of serum metabolites caused by DN.

Kidney tea alleviated ferroptosis in the kidneys of DN mice
Based on the results of untargeted metabolomics, we further investigated the roles of kidney tea in the regulation of ferroptosis pathway.Electron microscope analysis revealed destruction of mitochondrial cristae in renal tissues of DN mice, while high and medium doses of kidney tea significantly attenuated mitochondrial damage (Figure 5A).SOD is a key intracellular antioxidant system, whereas MDA and 4-HNE are indicators of lipid peroxidation.Therefore, the relationship between ferroptosis and DN was further explored by measuring SOD, MDA and 4-HNE levels.The Mod group had lower activity of SOD as well as higher levels of MDA and 4-HNE than the Con group (Figures 5B-D).These findings suggested the presence of ferroptosis in the kidneys of DN mice.Of note, these indicators were ameliorated after kidney tea intervention.Next, we determined the expression of the ferroptosis-related proteins acyl-CoA synthetase long-chain 4 (ACSL4), glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and nuclear receptor coactivator 4 (NCOA4) in renal tissues.The mRNA expression of NCOA4 and ACSL4 was significantly increased in DN mice compared to normal mice, while kidney tea could decreased their expression (Figures 5E,G).The mRNA expression of FTH1 and GPX4 was significantly decreased in DN mice, which was reversed by kidney tea administration (Figures 5F,H).Western blot was further utilized to detect the expression of these proteins, and the results were consistent with qRT-PCR (Figures 5I-M).In conclusion, these data indicated that ferroptosis contributed to DN development, and that kidney tea played a therapeutic role in DN by improving ferroptosis in the kidneys.

Correlations between gut microbiota, metabolites and host phenotypes
Spearman correlation analysis was conducted to determine the potential relationships between differential bacterial genera, altered metabolites, and host phenotypes (Figure 6).Our results found that Corynebacterium and Akkermansia were positively correlated with serotonin but negatively correlated with isopentenyl pyrophosphate and N-alpha-acetyl-l-ornithine.Meanwhile, Bacteroides was significantly and negatively correlated with serotonin.In addition, Prevotellaceae_UCG-001, Corynebacterium, Akkermansia, and Muribaculaceae were positively correlated with renal injury indicators (Scr, BUN, NAG and NGAL), pro-inflammatory cytokines (IL-6 and TNF-α) and ferroptosis markers (MDA and 4-HNE), but negatively correlated with SOD and IL-10.On the contrary, Blautia and Bacteroides had an opposite relationships with these phenotypes.Altogether, these data supported that some key gut microbiota might be related to host metabolites and DN development.

Discussion
Diabetes is a chronic metabolic disease characterized by hyperglycemia.In recent years, the prevalence of diabetes has been increasing with the changes in diet and lifestyle (Mangione et al., 2022).At the same time, diabetes-associated complications have become a health issue of great concern to the global community.Among them, DN is one of the most important microvascular complications.Kidney tea is widely used for DN treatment, while studies on the mechanism of action are still lacking.The present study revealed that kidney tea significantly reduced the levels of FBG in DN mice, which was consistent with other studies (Lokman et al., 2019;Nguyen et al., 2019).Importantly, kidney tea could also reduce the levels of Scr, BUN, urinary albumin, NGAL and NAG, suggesting that this herb showed good nephroprotective effects.Increasing evidence suggests that the inflammatory response plays a vital role in the development of DN (Shahzad et al., 2022;Wang et al., 2023;Huang et al., 2023).WNT1-inducible signaling pathway protein 1 (WISP1) activated inflammatory responses in the kidney through the nuclear factor-kappa B (NF-κB) pathway, which was characterized by the increased expression of CCL2, TNF-α and IL-6 (Wang et al., 2022).Recent studies have found that oxidative stress and inflammation promote tubulointerstitial fibrosis, whereas activation of exchange protein activated by cAMP (Epac) can attenuate the progression of DN by modulating the C/EBP-β/  Potential mechanisms of kidney tea in improving DN.
Frontiers in Pharmacology frontiersin.org10 SOCS3/STAT3 pathway (Yang et al., 2022).Our results showed that kidney tea intervention significantly reduced the levels of TNF-α and IL-6 and increased the levels of IL-10, suggesting that kidney tea attenuated inflammation in DN mice.Additionally, H&E, PAS and Masson staining confirmed that kidney tea ameliorated renal pathological damage.Collectively, these results suggest that kidney tea treatment can improve glucose metabolism and renal injury in DN mice.
Emerging evidence supports the involvement of the gut microbiota in the occurrence and progression of DN (Jiang et al., 2022;Zhang et al., 2023;Lu et al., 2023).Dysbiosis of gut microbiota resulted in increased acetate levels in DN rats, which promoted cholesterol homeostasis dysregulation and tubulointerstitial injury through activating G protein coupled receptor 43 (GPR43) (Hu et al., 2020).Furthermore, hyperactivation of complement C5 exacerbated renal dysfunction in db/db mice by promoting STAT3 expression and gut microbiota dysbiosis (Li et al., 2021).In the present research, kidney tea remodelled the gut microbiota in DN mice, manifested by an increase in the relative abundance of genera Blautia, Alloprevotella and Lachnospiraceae_NK4A136_group, as well as a decrease in the relative abundance of genera Muribaculaceae, Lachnoclostridium, Akkermansia, Corynebacterium and Prevotellaceae_UCG-001. Blautia are a group of anaerobic bacteria commonly considered to have probiotic properties.In DN mice with mild proteinuria, Blautia was negatively correlated with 24 h urinary protein levels (Li et al., 2020).Zhang et al. (2022) demonstrated that lycoperoside H could improve blood glucose and renal function in streptozotocin-induced DN rats by increasing the content of Blautia and Ruminococcaceae.Dysregulation of Alloprevotella is strongly associated with various pathological and physiological processes, such as atherosclerosis, ulcerative colitis and obesity (Kong et al., 2019;Liu et al., 2020;Wang et al., 2022).Compared with healthy individuals, both T2DM patients and DN patients have lower abundance of Alloprevotella and Megasphaera (Yang et al., 2022).Recent studies have found that resveratrol attenuates inflammation and kidney injury in db/db mice, and the mechanism is related to the elevated content of Alloprevotella, Bacteroides and Parabacteroides (Cai et al., 2020).Analogously, astaxanthin could protect against streptozotocin-induced DN by elevating the abundance of Lachnospiraceae_NK4A136_group (Ha et al., 2023).Consistent with these results, kidney tea might exert nephroprotective effects by enriching Blautia, Alloprevotella and Lachnospiraceae_NK4A136_group.
In addition to increasing the abundance of beneficial bacteria, kidney tea intervention inhibited the development of DN by decreasing the abundance of certain harmful bacteria.The diversity of the gut microbiota was lower in DN patients, and the relative abundance of Lachnoclostridium and Bacteroides was positively correlated with cholesterol levels (Chen et al., 2021).Moreover, Morchella esculenta polysaccharides might improve glycolipid metabolism and endotoxemia in T2DM mice by reducing the relative abundance of opportunistic bacteria such as Corynebacterium and Actinobacteria (Rehman et al., 2022).These data suggest that the reduced abundance of Lachnoclostridium and Corynebacterium may be the molecular mechanism by which kidney tea improves DN.Strikingly, it remains unknown whether Akkermansia is a friend or a foe in DN progression.Akkermansia was positively correlated with blood glucose, urinary albumin/creatinine ratio, and kidney/body weight ratio in DN mice, whereas Abelmoschus Manihot attenuated renal injury by reducing the abundance of Akkermansia (Shi et al., 2023).On the contrary, the content of Akkermansia was increased in ginsenoside compound K-treated db/db mice (Chen et al., 2022).Therefore, further studies are needed to elucidate the roles of Akkermansia in the pathogenesis of DN.
Serum metabolomics analysis revealed that kidney tea significantly reversed metabolite dysregulation.A total of 72 differential metabolites were identified between the Mod group and the KTH group.Of these, 41 metabolites were downregulated and 31 metabolites were upregulated after kidney tea supplementation.Previous studies have found that lower serum L-arginine levels and creatinine clearance, as well as higher postprandial glucose levels may be reliable predictors of microalbuminuria development in T2DM patients (Bariş et al., 2009).L-arginine and β-caryophyllene combination could attenuate renal fibrosis and inflammation in diabetic rats by inhibiting NF-κβ (Kumawat and Kaur, 2023).The higher initial levels of 3-hydroxybutyric acid indicated better control of HbA1c during drug treatment in newly diagnosed T2DM patients (Lee et al., 2023).Another study demonstrated that 3-hydroxybutyric acid could improve insulin resistance in diabetic mice by regulating hydroxycarboxylic acid receptor 2 (HCAR2) (Zhang et al., 2023).In addition, metabolomics analysis revealed that the levels of allantoin were higher in the kidneys and urine of DN rats compared to control rats (Liu et al., 2015).Similarly, choline content was significantly elevated in the plasma of diabetic patients and positively correlated with blood glucose levels (Li et al., 2023).In the present research, kidney tea intervention elevated the levels of arginine and 3-hydroxybutyric acid and decreased the levels of allantoin and choline, suggesting that kidney tea might prevent the development of DN by regulating these key metabolites.
Importantly, our results showed that kidney tea mainly participated in ferroptosis, arginine biosynthesis and mTOR signaling pathway.Ferroptosis, an iron-dependent form of regulated cell death, is considered to be closely associated with DN pathogenesis (Guo et al., 2023;Kim et al., 2023;Tsai et al., 2023).Attractively, emodin inhibited ferroptosis by promoting the expression of nuclear factor E2-related factor 2 (NRF2), thereby reducing reactive oxygen species (ROS) production and ameliorating renal injury in DN rats (Ji et al., 2023).Recent research has found that vitexin can improve ferroptosis-mediated DN by up-regulating solute carrier family 7 member 11 (SLC7A11) and GPX4 (Zhang et al., 2023).By electron microscope analysis, we confirmed that kidney tea attenuated mitochondrial damage in DN mice.Meanwhile, kidney tea significantly increased SOD activity and decreased MDA and 4-HNE levels, indicating that this herb attenuated lipid peroxidation in kidney tissues.ACSL4 is involved in the biosynthesis of polyunsaturated fatty acid (PUFA)-phospholipid in the cell membrane, which drives the operation of ferroptosis (Dai et al., 2023).Ferritin consists of two types of subunits FTH1 and ferritin light chain (FTL), and is primarily responsible for intracellular iron storage (Zhang et al., 2021).NCOA4 caused iron overload by mediating ferritin degradation, thereby promoting lipid peroxidation and ferroptosis (Jin et al., 2023).
Conversely, GPX4 is a key anti-ferroptosis factor that exerts its antioxidant effect by converting lipid hydroperoxides to lipid alcohols (Bersuker et al., 2019).Kidney tea treatment increased the expression of FTH1 and GPX4 and decreased the expression of ACSL4 and NCOA4 in the kidneys of DN mice.Collectively, these findings suggest that kidney tea may protect against the progression of DN by inhibiting ferroptosis.

Conclusion
In summary, our research demonstrates that kidney tea treatment attenuates glucose metabolism disorders, systemic inflammation, and renal injury in DN mice, and that the underlying mechanisms may be associated with the regulation of gut microbiota and metabolic profile (Figure 7).Meanwhile, kidney tea alleviates lipid peroxidation and ferroptosis in the kidneys by regulating the expression of NCOA4, FTH1, ACSL4 and GPX4.These findings provide a novel scientific rationale for the clinical application of kidney tea in DN.Future studies are needed to further clarify the active ingredients of kidney tea.Moreover, the specific relationship between gut microbiota and ferroptosis in the context of DN remains to be explored through fecal transplantation.

FIGURE 3
FIGURE 3 Kidney tea reshaped the gut microbiota in DN mice.(A) Principal coordinate analysis (PCoA) of the gut microbiota among all groups.(B, C) Relative abundance of the gut microbiota at the phylum and genus levels.(D-K) Relative abundance of eight key bacteria at the genus level.(L) Distribution histogram based on LDA score.*p < 0.05, **p < 0.01 compared with the Mod group.

FIGURE 4
FIGURE 4 Kidney tea reversed the serum metabolomics profile in DN mice.(A, B) Principal component analysis (PCA) score plots of all samples under positive and negative ion modes.(C) Heat map of the top 50 serum differential metabolites.(D) Enrichment analysis of differential metabolites between the Mod group and the KTH group.(E-J) Relative abundance of six differential metabolites.*p < 0.05, **p < 0.01, ***p < 0.001 compared with the Mod group.

FIGURE 6
FIGURE 6 Spearman correlation analysis of the relationship between gut microbiota, serum metabolites, and host phenotypes.Red indicates a positive correlation and green indicates a negative correlation.*p < 0.05, **p < 0.01, ***p < 0.001.

TABLE 1
Identification of polyphenols with relative percentages higher than 0.5% in kidney tea.