| Objective:The global prevalence of diabetes is increasing,and about 20%of diabetic patients may progress to diabetic nephropathy(DN).Despite significant benefits of the therapeutic measures of DN in slowing the progression of diabetic nephropathy,the occurrence of diabetic renal clinical events is still not reduced.In recent years,attention has been paid to the role of the gut microbiota and its metabolites in the development of diseases,and the gut-renal axis has gradually become one of focus of metabolic research.Dietary fiber is an important prebiotic that regulates composition and metabolism of the gut microbiota.And,the effect of dietary fiber supplementation on progression of disease has received more and more attention.However,the effect of inulin-type fructans(ITFs)on diabetic nephropathy remain unclear.This study aims to explore the protective mechanism of inulin-type fructans on diabetic nephropathy,and further improve the understanding of the gut-renal axis in the pathogenesis of diabetic nephropathy.Moreover,it provides new ideas and targets for the prevention and treatment of diabetic nephropathy.Methods:1)Eight-week-old db/db mice were used to establish an animal model of diabetic nephropathy.ITFs was added into drinking water to the mice for 12 weeks.The general biochemical indexes and renal biochemical indexes of each group were detected to evaluate the effects of ITFs on renal function.Staining of tissue were used to observe the pathological changes of kidney tissue.Changes in the levels of fibrosis-related proteins quantified the degree of renal fibrosis.2)For investigate the effects of ITFs on the composition characteristics of the gut microbiota,the fecal samples of mice in each group were collected for 16S rRNA microbial diversity sequencing.The serum and fecal samples of mice were collected to detected the levels of short-chain fatty acids in serum and fecal samples by gas chromatography mass spectrometry(GC-MC).The Pearson index correlation analysis was used to confirm the involvement of the gut microbiota and its metabolites in mediating the renal protection of ITFs.3)Antibiotics mixture was used to remove most of the gut microbiota in db/db mice by gavage.The intestinal ecological environment of germ-free mice was simulated to explore the role of ITFs in the progression of diabetic nephropathy after the removal of intestinal flora.Subsequently,fecal suspensions of the db/db mice treated with ITFs fecal microbiota transplantation to assess the changes of renal function,renal pathology and renal fibrosis after fecal transplantation.4)To clarify the role and mechanism of acetate,a metabolite of the gut microbiota,in mediating renal protection.In vitro,fibrosis of mouse mesangial cell line(SV40 MES13)induced by high glucose was used to simulate the model of diabetic nephropathy.The protein and transcription levels of fibrosis-related proteins were detected to quantify the effect of acetate on hyperglycemia-induced SV40 MES13 fibrosis.Sodium acetate was added into drinking water of mice to detect the effects of acetate supplementation on renal function,renal pathology and degree of renal fibrosis.The effects of acetate on mitochondrial function were evaluated by detecting the levels of mitochondrial respiratory chain related enzymes,mitochondrial membrane potential and reactive oxygen species.At the same time,the effects on glucose metabolism were evaluated by detecting the levels of glycotoxic metabolism-related enzymes and glycotoxic products.Results:1)ITFs supplementation decreased serum creatinine and blood urea nitrogen levels and urinary albumin creatinine ratio in db/db mice.ITFs reduced mesangial cell proliferation,basement membrane thickening,and significant extracellular matrix deposition in db/db mice,as well as glomerular deposition of fibronectin and type Ⅳcollagen,and up-regulated expression of fibrosis-related proteins in renal tissues.2)ITFs supplementation had no significant effect on the Alpha diversity analysis of fecal microflora in each group,but significantly changed the gut microbiota community structure of db/db mice.Supplementation with ITFs increased the abundance of beneficial bacteria such as Akkermansia,Mucispirillum,Candidatus Saccharimonas and Prevotella 2.ITFs increased acetate levels in stool and serum of db/db mice.And,ITFs-induced enrichment of Akkermansia and Candidatus Saccharimonas were closely related to the production of acetate.3)Antibiotic pretreatment and fecal microbiota transplantation further confirmed the key role of the gut microbiota in mediating renal protection of ITFs.After antibiotic pretreatment to deplete most of the endogenous microbiota,ITFs no longer had protective effects on renal function,renal pathology and fibrosis in db/db mice.Microbiota-depleted db/db recipient mice were reconstituted with the microbiota of ITFs-treated db/db mice by fecal microbiota transplantation,and the kidney protection effect was consistent with that of ITFs.ITFs did not significantly increase the concentration of acetate in serum and feces of the microbiota-depleted group,while microbiota transplantation had the same effect of enriching acetate content as supplementing ITFs.4)Acetate inhibited glomerular mesangial cell fibrosis induced by high glucose.The supplementation of acetate delayed the deterioration of renal function and improves the pathological injury and fibrosis of kidney in db/db mice.At the same time,acetate inhibited the production of intracellular reactive oxygen species and improved the dysfunction of mitochondria in renal tissue.The supplementation of acetate regulated the disorder of glucose metabolism and inhibited the accumulation of glycotoxic metabolites in the kidney.Conclusions:1)ITFs improved renal function,renal pathological injury and renal tissue fibrosis in db/db mice,and had beneficial effects on prevent the development of diabetic nephropathy;2)ITFs remodeled the gut microbiota of db/db mice,and increased the production of acetate in feces and serum by enrichment of bacteria closely related to shortchain fatty acids production such as Akkermansia and Candidatus Saccharimonas;3)The gut microbiota mediated the renoprotective effects of ITFs,and its metabolite acetate was positively correlated with the renal protective effect of ITFs.The gut-renal axis played an important role in the progression of diabetic nephropathy;4)Acetate,a metabolite of the gut microbiota,as a link molecule of the gut-renal axis,delayed the progression of diabetic nephropathy by inhibiting the accumulation of toxic glucose metabolites in renal tissue and improving mitochondrial function. |
