In the present study, we showed an obvious change in the richness of gut microbiota between DM patients and DN patients, and the altered bacterial community may play an important role in the progression of DN. Tao et al. also showed the similar shift of the richness of gut microbiota in DN patients. SCFAs such as acetate, propionate, and butyrate are the major microbial products and important energetic and signaling molecules. It was reported that serum SCFA levels were lower in CKD patients than healthy controls. In this study, the concentration of serum acetate, propionate, butyrate and total SCFAs were lower in DN group than those in NC group and DM group. The serum butyrate level was positively correlated with eGFR level, but negatively correlated with UACR level. It suggested that SCFAs may be a new therapy to delay the progression of DN.
The reduced level of serum butyrate was positively correlation with the decreased level of fecal butyrate in DM rats in our study. Yamamura et al. demonstrated that fecal SCFAs level was likely to be positively associated with serum SCFAs level, and the relative abundance of several SCFAs-producing bacteria were positively associated with fecal SCFAs even after adjusting for age and sex[20–22]. We speculated that the disordered gut microbiome and decreased certain SCFA-producing bacteria may result in the reduced serum SCFAs level in DN patients. Tao et al. showed that at the genus level SCFAs-producing bacteria Prevotella_9 decreased in DN group. In our study, the results revealed that DN patients had apparent dysbiosis in gut microbiota, especially the decreased relative abundance of Ruminococcaceae, Butyricicoccus and Lachnospiraceae, which were SCFAs- producing bacteria of the human gut in Eastern China. In addition, the relative abundance of butyrate-producing bacteria, Ruminococcaceae_UCG-014, was decreased obviously in DM rats compared with normal rats in our study.
In order to demonstrate the effects of SCFAs on DN, we selected sodium butyrate, one of the most biologically effective SCFAs from microbial fermentation in the gut, to treat DM rats. Although probiotic supplements can increase the butyrate level, they occasionally cause detrimental metabolic activities or produce host deleterious metabolites, and cause inappropriate immune responses and systemic infections . Herein, oral butyrate supplement was used to increase the serum butyrate level. After treatment with sodium butyrate, the DM rats showed reduced kidney damage in our study. Furthermore, sodium butyrate improved gut dysbiosis, promoting expansion of the genera Ruminococcaceae_UCG-014. Other reported studies also observed improvements in kidney injury in other models upon the butyrate treatment[24, 25] Li et al. revealed fiber promoted expansion of Prevotella and Bifidobacterium, which increased fecal and systemic SCFA concentrations and reduced expression of genes encoding inflammatory cytokines, chemokines, and fibrosis-promoting proteins in diabetic kidneys. The fecal microbial transplantation was shown to increase the level of fecal butyrate in DN mice, and also improved renal pathological injury. The In vitro studies also showed sodium butyrate played protective effects on podocytes, tubular cells, endothelial cells and mesangial cells under high glucose status.
Autophagy is a highly conserved cellular process to maintain intracellular homeostasis. Accumulated evidences have shown that activation of autophagy can prevent the progression of DN. In this study, our results showed that LC3 mRNA level and LC3BⅡ/Ⅰ ratio in DM group were significantly lower than those in control group, and there was little autophagosomes in renal tissue, which indicated that autophagy was inhibited in DM rats. This finding is consistent with the other study. Then, upon the treatment with sodium butyrate, LC3 mRNA level and LC3BⅡ/Ⅰ ratio, as well as the number of autophagosomes in DM + BU rats increased compared with DM rats. So, we firstly showed that sodium butyrate could trigger autophagy in kidneys of DM rats. Recently, it was reported that SCFAs could regulate autophagy in acute kidney injury by promoting the expression of Atg 7 in renal tubular epithelial cells to inhibit renal fibrosis and improve renal function. In other diseases, sodium butyrate has been found to induce Atg 5-dependent autophagy in mouse neuroendocrine STC-1 cells, which was mainly characterized by increasing expression of LC3-Ⅱ and decreasing expression of p62. Other studies also found that sodium butyrate promotes autophagy to inhibit tumor growth in colorectal cells [32, 33] .
We further investigated the mechanism of sodium butyrate in regulating autophagy in DM rats. The AMPK/mTOR pathway is the classic pathway to regulate autophagy. Butyrate was reported to activate AMPK phosphorylation to improve renal injury in CKD animals. Wang et al. proved that the AMPK/mTOR pathway mediated sodium butyrate induced autophagy in human bladder cancer cells. Our study found that sodium butyrate dramatically increased p-AMPK/AMPK, with AMPKα phosphorylation at Thr172, and downregulated p-mTOR/mTOR, with mTOR phosphorylation at Ser2448. These findings confirmed that the AMPK/mTOR pathway may mediate sodium butyrate induced autophagy in kidneys of DM rats.
Also, we observed that oral sodium butyrate supplement in DM rats decreased blood glucose level significantly after 8 weeks and 12 weeks. It was in coincided with previous studies[36, 37]. Gao et al showed that there was a significant and positive effect of sodium butyrate on glucose metabolites and insulin resistance. In a randomized double-blinded placebo-controlled trial, combination of butyrate and inulin significantly reduced fasting blood sugar and waist to hip ratio. A study also demonstrated that butyrate-producing bacteria Clostridium butyricum CGMCC0313.1 (CB0313.1) improved diabetic markers of fasting glucose, glucose tolerance and insulin tolerance. Butyrate-mediated glucagon like peptide-1 (GLP-1) secretion may be a major factor in the improvement of insulin sensitivity and diabetes. GLP-1 has been demonstrated to decrease hepatic gluconeogenesis and promote insulin secretion. The increased glucose transporter-4 (GLUT-4) level induced by butyrate in adipose tissue may be considered as one of the other factors to improve glucose metabolites. GLUT4, expressed in skeletal muscle and adipose tissue, is a member of glucose transporter protein family to uptake the glucose in tissues[42, 43]. Taken together, this may be an extra effect for controlling hyperglycemia to alleviate the progression of DN.
There were several limitations in our study. Firstly, the direct correlation between serum SCFAs and gut microbiota would not be analyzed in the participants, as they were not from the same cohort. Secondly, the lifestyles or diets were not analyzed due to a lack of records. So, the association between gut microbiota composition and lifestyles or diets needs to be explored in further studies. Thirdly, gene knockout mice, germ-free mice and fecal microbiota transplantation (FMT) were not used in the animal study. Herein further studies on the underlying mechanism and interactions are needed.
In conclusion, we demonstrated the changes in composition of the gut microbiota, especially the decrease in SCFAs-producing bacteria in DN patients. In addition, we showed that serum concentrations of SCFAs were low in DN patients, and oral butyrate supplementation may improve kidney injury in DM rats, possibly by increasing autophagy via activating AMPK/mTOR pathway (Fig. 5). These findings provided further understanding of gut-kidney axis in diabetic nephropathy and may provide a new promising strategy.