Dietary Astaxanthin Supplementation could regulate the Gut-Kidney axis to protect against Kidney Impairment in Type 2 Diabetes mice

doi:10.21203/rs.3.rs-1611723/v1

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Research Article

Dietary Astaxanthin Supplementation could regulate the Gut-Kidney axis to protect against Kidney Impairment in Type 2 Diabetes mice

https://doi.org/10.21203/rs.3.rs-1611723/v1

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posted 18 May, 2022

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Background: There remains unclear whether the reshaped intestinal microecology influenced by dietary astaxanthin supplementation could protect against type 2 diabetes caused kidney disease. In this study, we investigated the benefits of dietary astaxanthin supplementation on modulating gut microbiota and on interactions between diet, gut microbiota and kidneys using a rodent model.

Method: Twenty-five out of 30 C57BL/6J male mice were successfully modeled by a high fat diet (60%) and then intraperitoneally injected by streptozotocin (35mg/kg) and randomly assign to either the four astaxanthin groups (12, 24, 48, and 96mg/kg, n=5 for each group) or the diabetes nephropathy group (n=5). The normal control group (n=5) received a vehicle diet and intraperitoneally injected the equivalent dose of citrate buffer.

Results: After a 12-week dietary astaxanthin supplementation at four different doses of 12, 24, 48, and 96mg/kg, there presented a dose-dependent relationship on optimizing the ratio of Actinobacteria to Bacteriodetes. Except for 12mg/kg, the astaxanthin at other doses altered the composition of microbial communities to varying degrees. In addition, astaxanthin at 48 and 98mg/kg largely maintain the balance of gut microbiota by suppressing the over-reproduction of the genera such as Clostridium_sensu_stricto_1, Coriobacteriaceae_UCG-002, Romboutsia, and Lactococcus, and boosting the scarce genus of g__Lachnospiraceae_NK4A136_group, further ameliorating renal pathological development. Concomitantly, astaxanthin also reduced the serum creatinine, and astaxanthin at 96mg/kg significantly decreased the urine albumin-to-creatinine ratio.

Conclusion: Astaxanthin could reshape intestinal flora, mildly inhibit inflammation and oxidative stress to protect against type 2 diabetes-provoked kidney impairment. The study may provide evidence of the effects of astaxanthin at 48 and 96mg/kg on shifting gut microbiota and preventing type 2 diabetes kidney disease.

dietary astaxanthin supplementation

gut microbiota

inflammation

oxidative stress

type 2 diabetes kidney disease

Type 2 diabetes is a serious public health threat. According to the 10th edition of International Diabetes Federation (IDF) diabetes Atlas, there are about 537 million people living with diabetes, and the amount is predicted to arise to 643 million (11.3%) by 2030 and to 783 million (12.2%) by 2045, over 90% percent among which were people with type 2 diabetes (T2DM)[1]. Impressively, approximately half of the adults with type 2 diabetes would develop chronic kidney disease [2]. Diabetes kidney disease is featured with glomerular hyperfiltration, proteinuria, the mesangial hypertrophy, mesangial matrix accumulation, renal hypertrophy and later glomerulosclerosis [3–4]. Its pathogenesis also includes the renal hemodynamics abnormalities, the renin-angiotensin-aldosterone system, oxidative stress, inflammatory responses and fibrosis [5]. Occurrence and severity of chronic kidney disease increase the risks for adverse health outcomes in diabetic patients, including frailty, worsen quality of life, end-stage renal disease, progressive end-organ damage at other sites and premature mortality[2]. Accordingly, the prevention and management result in substantial immediate and long-term health care expenditures each year in both developed countries and developing economies [6].

Astaxanthin has attracted considerable interests due to its potential anti-inflammatory, antioxidative, and antidiabetic properties, as well as its nephroprotective effects [7]. Astaxanthin produced from Heamatocaccus pluvialis is a main source for human consumption, as well as for dietary supplement in humans and animals [8]. Previous studies have demonstrated the effect of astaxanthin on mitigating nephropathy in diabetes rodent models by inhibiting the urinary albumin, decreasing the 8-hydroxydeoxyguanosine level in both the urine and the glomeruli [9], and alleviating oxidative damage and preventing pathological changes [10], as well as reducing renal fibronectin and collagen IV accumulation [11]. But, the bioaccessibility of astaxanthin was less than 50% and 10% in the normal and the obese body, respectively. Therefore, it is quite necessary to boost the possibility of the interaction between astaxanthin and gut microbiota [12]. Previous studies verified that diabetes kidney disease links to gut dysbiosis [13–14], featured by a bidirectional interaction between the gut microbiota and the kidneys, therefore, the deterioration of this crosstalk facilitates CKD development [15]. Diabetes-triggered dysbiosis of the microbial ecology generates substantial harmful substances, such as excessive uremic toxins and lipopolysaccharide (LPS), destroy intestinal mucosal immune responses and form the "leaky gut", which contributed to the entry of LPS into the bloodstream, and further resulted in chronic inflammatory responses, hemodynamic changes, energy imbalance, glucose and lipid metabolism disorder and vascular endothelial damage, and finally accelerated the progression of kidney disease [16–17].

Recently, astaxanthin changes the gut microbial composition which is associated with inflammation and metabolic homeostasis [18]. Astaxanthin plays beneficial roles in bridging interactions between the host and intestinal flora by the gut-liver axis. An earlier study demonstrated that astaxanthin protected against liver impairment in an alcoholic fatty liver mice model by balancing the gut microbiota via decreasing the genera Butyricimonas, Bilophila, and Parabacteroides, and increasing species from Verrucomicrobia and Akkermansia [19]. Later on, astaxanthin reduced obesity in the obese mice by optimizing the ratio of Firmicutes to Bacteroides and inhibiting the abundance of obesity-related pathogenic microbiota to modulate lipid metabolism in both liver tissue and serum levels [20–21]. However, the effects of astaxanthin from Haematococcus pluvialis on gut microbiota and renal pathogenetic processes in type 2 diabetes mice have not been elucidated so far and need further investigations.

Animals

Six-week-old C57BL/6J male mice (16-20g) with specific pathogen free (SPF) were purchased from Pengxin Bier Laboratory Animal Sales Co., LTD. Chongqing (SCXK 2019-0008). All mice were fed and watered freely and housed separately in a controlled room with a 12-h light/dark cycle, an ambient temperature of 20℃±2℃ and a relative humidity of 55%±5%. Animal experiments were carried on and approved by the Research Ethics Committee of Chongqing Population and Family Plan Science and Technology Research Institute (DWLL2021-001), and abided by the Guide for the Care and Use of Laboratory Animal published by the Ministry of Health of China.

Experimental Protocols and Groups

Thirty mice were assigned to a normal control group (n = 5) and type 2 diabetes group (n = 25). The normal control group were fed with a vehicle diet while the type 2 diabetes model mice were fed with a 60% high fat diet (60% HFD, research diet #d12492) for 3 three weeks according to the previous protocol [22]. During the fourth week, the freshly prepared streptozotocin (STZ) of 35 mg/kg was injected intraperitoneally for three consecutive days to establish the type 2 diabetes model, while an equal volume of citrate buffer was administered for the normal group mice. Afterwards, the type 2 diabetes mice were continued to fed with 60% HFD for four weeks, and the fasting blood glucose (FBG) levels were tested weekly after intraperitoneal injection using ACCU-CHEK Active. The type 2 diabetes mouse was considered eligible if the FBG ≥ 11.1mmol/L a week after the SZT-intraperitoneal injection, as well as either keeping the FBG at a high level or even rising for the following four weeks.

Besides the normal control group N (n = 5), the diabetes mice were randomly divided into five groups: astaxanthin 12 mg/kg (group A, n = 5), astaxanthin 24 mg/kg (group B, n = 5), astaxanthin 48 mg/kg (group C, n = 5), astaxanthin 96 mg/kg (group D, n = 5), and diabetes nephropathy (group E, n = 5). The astaxanthin was purchased from Kunming Biogenic Co., Ltd, China (HPLC ≥ 2.5%, batch number: CWS201125). The custom feed was made of the 60% HFD and four different doses of astaxanthin (12, 24, 48, and 96mg/kg/d). The daily astaxanthin intake could be achieved by consuming 2 g of custom feed per day. After 12 weeks, all the mice were weighted and sacrificed after the intraperitoneal anesthesia of 2.5% chloral hydrate. Urine and stools samples were obtained one or two days before they were sacrificed. Blood and kidneys were collected for subsequent tests.

Biochemical Assays

Serum concentrations of urea and creatinine were examined using a Chemray 800 automatic biochemical analyzer (Wuhan Servicebio Technology Co., Ltd., China). Levels of urine albumin and creatinine, serum creatinine (Cr), and blood urea nitrogen (BUN), concentrations of serum inflammatory indicators [Interleukin 10 (IL-10), Interleukin 1β (IL-1β), Interleukin 6 (IL-6), tumor necrosis factor α (TNFα), and Lipopolysaccharides (LPS)], and oxidative stress [reactive oxygen species (ROS) and malondialdehyde (MDA)] in the kidney were determined using mouse enzyme-linked immunosorbent assay kits (ELISA, Shanghai Enzyme-linked Biotechnology Co., Ltd, China). The coefficient of variation of inter-intra tests was less than 10%. Samples were determined in duplicate. Urine albumin-creatinine ratio (ACR) was determined by the urine albumin to urine creatinine ratio.

Histopathology

Formalin-fixed and paraffin-embedded kidney tissue samples were stained with periodic acid schiff (PAS) and Masson’s trichrome for histological analyses. Renal histopathological staining and analyses were performed by Wuhan servicebio technology CO., TLD., China.

Quantitative real-time PCR (RT-PCR)

Total RNAs of mice kidney tissue samples were isolated using RNA Easy Fast Tissue/Cell kits according to the manufacturer’s protocol (Tiangen Biotech Corp., China). First-strand cDNAs were synthesized using corresponding kits provided by Tiangen Biotech Corp. China, and PCR was carried out using SYBR Green qPCR kits (Invitrogen, USA), as the fluorescence indicator, according to the manufacturer’s instructions. Primer sequences of mRNAs were synthesized by RiboBio Co. Ltd. China (Supplementary Table S1). Samples were assayed in triplicate. The mRNA levels of target genes were normalized to the GAPDH mRNA levels.

Gut microbiome profiling

Microbial community genomic DNA was extracted from mice stools samples at the 12-week astaxanthin supplementation using the PowerFecal DNA kit (Qiagen, Germany) according to manufacturer’s instructions. To amplify the V3–V4 region of the 16S rRNA gene for Illumina deep sequencing, the primers 341F: 5’-CCTAYGGGRBGCASCAG-3’ and 806R: 5’-GGACTACNNGGGTATCTAAT-3’ were employed for the abundance estimate of total bacteria. Bioinformatics detection of intestinal flora genes was performed by Majorbio BioPharm Technology Co. (Shanghai, China).

Statistical Analysis

Alpha (α) diversity of Shannon, and abundance-based coverage estimator (ACE), for the complexity of species diversity for an individual sample, was calculated with mothur software (v.1.30.2) and displayed with the R software (V3.3.1). Beta (β) diversity, for evaluating differences among samples in species complexity, was calculated by the Quantitative Insights Into Microbial Ecology (QIIME v1.91) software. Beta diversity analysis was visualized by non-metric multidimensional scaling ordination (NMDS) based on Bray–Curtis dissimilarity matrices. The significance of microbiota community differentiation among groups was calculated by the method of the permutation test and ANOSIM (analysis of similarities) using the vegan package of R software. Each group distribution of gut microbiota communities and their potential correlations with clinical factors of urine albumin-creatinine ratio (ACR), serum creatinine (Cr), and blood urea nitrogen (BUN), fasting blood glucose (FBG), and body weight (Wt) were determined by redundancy analysis/canonical correspondence analysis (RDA/CCA), using the vegan package of R software. Spearman correlation were further employed to present the correlations of the relative abundance of the gut microbial community with the clinical parameters of renal function, FBG, and Wt. Taxa abundances at different taxonomies were statistically compared among groups by Metastates and visualized as box plots. Microbial functions were predicted utilizing PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), and then functional categorization was performed according to KEGG (http://www.genome.jp/kegg/; Supplementary, S8 & S9).

Quantitative results were expressed as the mean (M) ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by LSD or Dunnett’s post hoc test, and covariance (ANCOVA) analysis was also used to test the changes of FBG levels across different time points followed by Bonferroni for pairwise comparison, using SPSS 19.0. p < 0.05 was considered statistically significant.

Effects of Astaxanthin Supplementation on Modifying the Body Weight and the Fasting Blood Sugar in T2DM Mice

There was non-significant difference in the body weight before and after the three-month astaxanthin dietary supplementation among all groups (Fig. 1A). Also, there wasn’t drastic body weight gain or loss among groups. Astaxanthin might play a certain role in decreasing FBG level, because the FBG levels in groups C and D were marginally decreased after 12-week astaxanthin supplementation (p = 0.079, p = 0.05, respectively) by controlling the covariance of FBG levels at the baseline before astaxanthin supplementation (Fig. 1B), although the FBG levels didn’t show statistical differences at 4- and 8-week astaxanthin supplementation.

Effects of Astaxanthin Supplementation on Shifting the Profiles of Gut Microbiota in T2DM mice

High-throughput 16S rRNA gene sequencing generated a total of 1,614,167 good-quality sequences from 20 samples (Supplementary S1-S3). Alpha-diversity analysis of ACE showed statistically significant in groups N and B, Shannon indexes displayed significant differences in the groups B, D, and N (Fig. 2A-B). The community taxonomy composition of bacterial in groups on operational taxonomic unit (OTU) level was revealed by NMDS ordination (Fig. 2C) (Stress = 0.128, R = 0.5994, p = 0.001). Microbial composition in the groups B, C, and D was displayed in the middle between the control group N and the groups A and E (Supplementary S4-S9). According to the ANOSIM analysis, bacterial communities in genus level presented statistical differences among all groups (R = 0.595, p = 0.001). Compared with the group E, both the groups B and D shown significant changes (R = 0.482, p = 0.023; R = 0.630, p = 0.023, respectively), and the group C presented marginal differences (R = 0.667, p = 0.098); however, the group A remained no statistical significance (R = 0.111, p = 0.298; Fig. 2D).

At 12-week astaxanthin supplementation, the bacterial phyla proportion of Firmicutes, Bacteroidetes, Actinobacteria, ratio of Firmicutes/Bacteroidetes (F/B ratio), and ratio of Actinobacteria/Bacteroidetes (A/B ratio) were altered differently in both the diabetes nephropathy group E and the astaxanthin groups. Compared with the norrmal control group N, the abundance of Actinobacteria and Firmicutes in the group E was statistically increased by 23.2% and 18.7%, respectively, whereas the Bacteroidetes abundance was dramatically decreased by 41.62%. Therefore, the ratio of phylum Firmicutes to Bacteroidetes (F/B) was significantly increased from 0.66 (group N) to 3.35 (group E), and the ratio of phylum Actinobacteria to Bacteroidetes (A/B) was statistically upregulated from 0.0004 (group N) to 1.34 (group E). The 12-week astaxanthin supplementation could not only drastically decrease the abundance of Actinobacteria, but slightly increase the Bacteroidetes abundance as well. It presented a dose-dependent relationship in shrinking both the F/B ratio from 6.47 (group A) to 2.51 (group D) and the A/B ratio from 1.51 (group A) to 0.08 (group D). The abundance of Actinobacteria in the astaxanthin groups B, C, and D was statistically decreased compared with the group E, and exhibited a sharp decline from 16.09% (group A) to 2.13% (group D) (Fig. 2EF). At genus level, levels of Coriobacteriaceae_UCG_002, Romboutsia, Clostridium_sensu_stricto_1, and Turicibacter were increased markedly, whereas levels of Lanchospiraceae_NK4A136_group, Roseburia, and norank_f_Muribaculaceace were dramatically decreased in the group E compared with the group N. Still, levels of Prevotellaceae_UCG-001, Ruminococcus, Eubacterium_xylanophilum_group were almost depletion, whereas Lactococcus was detected and kept at a high level only in the group E. Astaxanthin supplementation partially reversed these abnormal changes. Firstly, Lactococcus was completely depleted, and Coriobacteriaceae_UCG_002 and Turicibacter were significantly decreased in the groups B, C, and D. Secondly, Romboutsia in the group C and Clostridium_sensu_stricto_1 in the group D were significantly decreased, whereas Lanchospiraceae_NK4A136_group and Oscipplospia were significantly increased in the group D. However, the impacts of astaxanthin were little on the levels of Roseburia, Bifidobacterium, and Blautia (Fig. 2GH).

The RAD/CCA analysis at genus level was used to uncover the potential correlations between the distribution of bacterial assemblages in each group and the clinical factors, such as renal function indicators (ACR, Cr, and BUN), Wt and FBG. It was found that FBG (p = 0.006), and ACR (p = 0.025) appeared to be the most significant among the investigated clinical factors (Fig. 3A). Spearman’s correlation heatmap presented the associations between the relative richness of the intestinal microbial community at the genus level and the parameters of renal function (ACR, serum Cr, BUN), fasting blood glucose, and body weight. At genus levels, Lachnospiraceae_NK4A136_group, Erysipelatoclostridium, norank_f_norank_o_Clostridia_vadinBB60_group, and norank_f_Muribaculaceae were negatively associated with the levels of FBG and ACR. Conversely, Coriobacteriaceae_UCG_002, Clostridium_sensu_stricto_1, and Lactococcus were positively correlated with the levels of FBG and ACR (Fig. 3B).

Effects of Astaxanthin Supplementation on Renal Inflammation and Oxidative Stress in the T2DM mice

After 12 weeks dietary astaxanthin supplementation, the ACR in the group E was significantly higher than in the groups N (p = 0.015), but astaxanthin at 96 mg/kg (the group D) could statistically decrease the level of ACR (p = 0.045). There was no significant difference in the level of serum BUN in all groups. However, the level of serum Cr in the groups A (p = 0.008), C (p = 0.026), and D (p = 0.001) was decreased statistically, and there was a marginal decrease in the group B (P = 0.061), compared with the group E (Fig. 4A). Again, according to the PAS and Masson’s staining results, the histopathological features of the kidney tissue presented mild to moderate glomerular mesangial cell hypertrophy, and many vacuoles in the renal tubule epithelial cells with swelling in the group E (Fig. 4B). Unfortunately, the group A presented the similar renal pathological changes as the group E, however, it shown a smaller number of renal tubules had balloon and vacuolar degeneration in the group B, and both the groups C and D presented comparatively fewer amount of degenerated vacuoles of renal tubules.

To evaluate the effects of astaxanthin on kidneys oxidative stress and inflammation in vivo, mRNAs of sirtuin 1 (Sirt1), peroxisome proliferator-activated receptor-γ coactivator (PGC-1α), nuclear transcriptional factor kappa B p65 (NFkBp65), oxidation resistance 1 (OXR1), superoxide dismutase 1 (SOD1), glutathione peroxidase (GSH-Px), Catalase (CAT), and concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA) in renal tissue, as well as serum inflammatory factors were tested (Fig. 5A). Compared with the group E (diabetes nephropathy), the Sirt 1 mRNA in the astaxanthin groups C and D was significantly increased by 55%, 88%, respectively. PGC-1α was statistically increased in the group D (p < 0.01), and marginally increased in the groups B and C (p = 0.054, p = 0.05). Moreover, NFkB p65 in the group C was statistically decreased (p < 0.01) and marginally decreased in the group D (p = 0.062). Furthermore, the ROS level in kidneys was significantly increased, whereas the anti-oxidative indicators of OXR1, SOD1, GSH-Px, CAT mRNAs were drastically decreased in the group E. However, astaxanthin, especially in the groups C and D, reversed the phenomenon by reducing ROS and strengthening the anti-oxidative indicators of OXR1, SOD1, GSH-Px, CAT mRNAs in kidneys. Interestingly, the level of ROS was significantly higher in the group A than that in the group E, and the MDA level presented a tendency of decline in the groups B, C, and D, but remained no significance.

Additionally, astaxanthin could mildly strengthen anti-inflammation by suppressing serum inflammatory cytokines after 12-week dietary supplementation (Fig. 5B). Astaxanthin at 48mg/kg significantly increased the serum level of Interleukin 10 (IL-10) by 27.52% compared with the group E. Again, astaxanthin at 48 and 96mg/kg statistically decreased Interleukin 6 (IL-6), tumor necrosis factor α (TNFα), and Lipopolysaccharides (LPS) by 28.773% and 36.34%, 22.75% and 29.94%, 19.62% and 29.89% compared with the group E, respectively, yet astaxanthin could not decrease the serum level of Interleukin 1β (IL-1β). Astaxanthin at 12 and 24mg/kg might play some effects on anti-inflammation but the roles were too subtle to be distinguished. To sum up, these data indicated that dietary astaxanthin supplementation could inhibit inflammation and oxidative stress in kidneys, thereby playing beneficial roles in delaying hyperglycemia-provoked renal impairment.

In the present study, we tested the effects of various doses of astaxanthin on kidney protection by modulating gut microbiota in male type 2 diabetes mice. The study exhibited an interplay between diet, gut microbiota, and kidneys. Our major findings are as follows: 1) dietary astaxanthin supplementation could bridge the gaps between gut flora and kidneys; 2) dietary astaxanthin supplementation at 24, 48, and 96mg/kg could shift the composition of microbiota communities to varying degrees in the type 2 diabetes mice; 3) dietary astaxanthin supplementation could mildly alleviate renal oxidative and inflammatory damages by partially boosting the beneficial microbiota and inhibiting the detrimental flora, thereby protecting renal functions in type 2 diabetes mice.

In line with a previous study [23], long-term high-fat-diet and hyperglycemia declined the total intestinal microbial abundance and diversity and resulted in gut dysbiosis. Apart from 12mg/kg, astaxanthin at 24, 48, and 96mg/kg for 12 weeks could impact on intestinal flora, evidenced as the decrease of the ratio of Actinobacteria/Bacteroidetes (A/B) at phylum level. However, the ratio of Firmicutes/Bacteroidetes (F/B) wasn’t significantly changed by astaxanthin. The ratio of F/B determines the body's absorption of calories in food to control the body weight gain or loss [24], and the little difference in the F/B ratio among groups in our study might explain the little difference of the body weight among groups. None significance of the body weight gain was in the diabetes nephropathy group after 12 weeks compared with the baseline, the possibly reason might be the hyperglycemia-provoked renal functional degradation of filtration and excretion [25].

Astaxanthin could modulate several intestinal bacteria genera so as to indirectly suppress inflammation. Coriobacteriaceae_UCG-002, belonging to the Actinobacteria phylum [26], is potentially associated with inflammatory responses [27–28]. Our study found that Coriobacteriaceae_UCG-002 and Lactococcus were significantly increased in the diabetes nephropathy group and positively correlated with FBG and ACR, yet they were significantly suppressed by astaxanthin at 24, 48, 96mg/kg. Astaxanthin could effectively inhibit the level of Clostridium_sensu_stricto_1, which was testified as a potential pathological marker and closely correlated with the upregulation of the lipopolysaccharide level [29]. In this study, there were strong positive correlations between Clostridium_sensu_stricto_1 and FBG and ACR, and its level was significantly decreased by astaxanthin. Additionally, astaxanthin at 96mg/kg significantly increased the level of Lachnospiraceae_NK4A136_group, which was statistically decreased in the group E and negatively associated with FBG and ACR. The previous studies demonstrated that Lachnospiraceae_NK4A136_group, as a potential butyrate-producer associated with metabolic fitness and gut homeostasis, can promote the repair of the intestinal mucous and has an anti-inflammatory property [30–31].

Astaxanthin could also shift several intestinal bacteria genera which might be closely associated with kidney diseases and glucose metabolism. We found the upregulation of Turicibacter, and Bifidobacterium, and the downregulation of Lactobacillus, unclassified Ruminococcaceae, and Oscillospira genera in the diabetes nephropathy group. It has been reported that the changes of these genera is closely associated with inflammation and chronic kidney disease, as well as renal failure [32]. Astaxanthin could decrease the level of Bifidobacterium, and Turicibacter, and increase the levels of Oscillospira, and unclassified Ruminococcaceae. Additionally, the increase in Roseburia level by astaxanthin is a beneficial microbe of producing butyrate, and negatively correlated with chronic kidney disease [33] and glucose intolerance [34]. Moreover, Romboutsia was positively correlated with ACR and was significantly increased in the diabetes nephropathy group, but was decreased by astaxanthin at 48mg/kg. Romboutsia belongs to the family Lachnospiraceae, which may suppress the growth of short fat chain acid (SCFA)-producing bacteria, thereby contributing to the development of diet-induced obesity and diabetes in obese mice [35–36]. Actually, astaxanthin couldn’t reverse the upregulated levels of Faecalibaculum and Blautia in the diabetes nephropathy group. The former may impair the gut barrier due to its pro-inflammatory property [37], and the latter was positively correlated with type 2 diabetes in the study.

Above all, we speculated that the upregulated genera of Clostridium_sensu_stricto_1, Coriobacteriaceae_UCG-002, and Lactococcus, and the downregulated genus of g__Lachnospiraceae_NK4A136_group might be implicated in the developing type 2 diabetes and related to kidneys damage. However, astaxanthin could impede the destruction of the gut barrier and reverse the metabolism of intestinal microbiota by partially modulating the balance of gut flora, subsequently, activating a variety of signaling pathways and initiating anti-inflammatory responses and anti-oxidation. As a previous study demonstrated that increased intestinal permeability could amplify the entry of LPS into the portal circulation, which then activated downstream inflammatory signaling pathways through TLR4 activation, such as the NFkB signaling pathway [38]. In our study, diabetes-provoked imbalance of gut microbiome was accompanied with the increase in oxidative stress, serum levels of LPS and proinflammatory cytokines, such as TNFα, and IL-6. Astaxanthin could reverse this phenomenon and downregulate the NFkBp65 mRNA by activating the Sirt 1/PGC-1α mRNAs in kidneys. The finding was consistent with a former study that the Sirt1/PGC-1α/NFκB p65 pathway was closely correlated with oxidative and inflammatory responses [39].

Modulating the gut microbiota may provide an additional strategy to enhance the treatment of T2DM and diabetes kidney disease [40]. In this study, we testified the effects of astaxanthin on restoring the balance of gut microbiota and protecting against type 2 diabetes kidney disease. Dietary astaxanthin supplementation at 48 and 96mg/kg could exhibit beneficial interactions between diet, gut microbiota, and kidney responses. Furthermore, astaxanthin activated the Sirt1/PGC-1α/NF-kB p65 pathway to prevent kidneys from inflammation and oxidative injuries, and subsequently improve the renal function by decreasing the urine albumin-to-creatinine ratio and serum creatinine. Our study provided some new insights into the benefits of astaxanthin from the perspective of gut microbiota and affirmed the effectiveness of astaxanthin on kidney protection in a STZ-induced type 2 diabetes mice model. Next, the effectiveness of dietary astaxanthin on modulating gut microbiota in patients with type 2diabetes should be further studied.

Ethics Approval

Animal experiments were carried on and approved by the Research Ethics Committee of Chongqing Population and Family Plan Science and Technology Research Institute (DWLL2021-001).

Availability of Data and Materials

The datasets generated for this study are available on request to the corresponding author.

Competing Interests

All authors declare no competing interests.

Funding

This work was supported by Chongqing Municipal Natural Science Foundation, Grant/Award Number: cstc2019jcyj-msxmX0417.

Author’s Contributions

Conceptualization, Research Design, MH, CJL, YL; Methodology, MH, YHY, MZW; Data curation, MH, TG, CJL, YL;Supervision, CJL,YL;Writing- original draft, MH, CJL,YHY. Writing- Reviewing and Editing, MH, CJL, YHY, TG, MZW, ZYC, YL. All the authors critically revised the manuscript, and approved the final manuscript. All the authors have agreed to authorship and order of authorship for this manuscript, and have the appropriate permissions and rights to the reported data.

Acknowledgement

The authors acknowledged professor Lian Li for the maintenance of animal houses while we housing the mice.

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No competing interests reported.

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posted 18 May, 2022

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Dietary Astaxanthin Supplementation could regulate the Gut-Kidney axis to protect against Kidney Impairment in Type 2 Diabetes mice

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Abstract

Figures

Introduction

Materials And Methods

Animals

Experimental Protocols and Groups

Biochemical Assays

Histopathology

Quantitative real-time PCR (RT-PCR)

Gut microbiome profiling

Statistical Analysis

Results

Effects of Astaxanthin Supplementation on Shifting the Profiles of Gut Microbiota in T2DM mice

Effects of Astaxanthin Supplementation on Renal Inflammation and Oxidative Stress in the T2DM mice

Discussion

Conclusion

Declarations

References

Competing Interests

Supplementary Files

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