Study On The Effect And Mechanism Of Gut Microbiota In Empagliflozin Ameliorating Diabetic Nephropathy

Objective:Through empagliflozin（EMPA）intervention on diabetic nephropathy（DN）model mice induced by high fat diet（HFD）and streptozotocin（STZ）,from the point of view of gut microbiota,we aimed to illustrate the effect and mechanism of EMPA in ameliorating DN.Moreover,on the basis of DN model,a pseudo-germfree mouse model treated with broad-spectrum antibiotics（ABX）was constructed to observe whether EMPA can still play its original effect effectively in the state of gut microbiota exhaustion,and to verify the role of gut microbiota in the amelioration of DN by EMPA,providing a novel research direction and therapeutic target for the mechanism study of EMPA in the treatment of DN.Methods:1.HFD combined with STZ were utilized to establish DN model.C57BL/6J mice were divided into three groups by simple randomization:normal control group（Normal food diet,NFD）,DN model group（HFD/STZ）and EMPA therapeutic group（HFD/STZ+EMPA）,with six mice in each group.NFD group was fed with 10%fat control diet,while HFD/STZ group and HFD/STZ+EMPA group were fed with 60%fat high fat diets until euthanasia at the 13th week.At the 4th week,the DN model was established by intraperitoneal injection of STZ(50 mg·kg^-1·d^-1)for five consecutive days.At the 6th week,mice were fasted for 12 h at night the day before and fasting blood glucose（FBG）was measured.From the 9th week,HFD/STZ+EMPA group was administrated EMPA（10 mg/kg）gavage daily,and NFD and HFD/STZ groups were given equal volumes of sterile water gavage daily,for four consecutive weeks.Weekly weight measurement was performed from 0 to 13 weeks.Oral glucose tolerance test（OGTT）was conducted two days before mice euthanasia,and fresh feces were collected for two consecutive days for sequencing of the 16S ribosomal RNA（16S r RNA）V3-V4 region,detecting of short chain fatty acids（SCFAs）by gas chromatography-mass spectrometry（GC-MS）and lipopolysacch-aride（LPS）by enzyme-linked immunosorbent assay（ELISA）.One day before euthanasia,urine was collected for detection of albumin creatinine ratio（ACR）.Before euthanasia,blood samples were collected for detection of serum LPS by ELISA.After euthanasia,kidney and colon tissues were collected to observe the pathomorphological changes by renal periodic acid schiff（PAS）staining and Masson（Masson）staining,and colon hematoxylin-eosin（HE）staining,alcian blue periodic acid schiff（AB-PAS）staining and immunofluorescence staining.2.On the basis of DN model,broad-spectrum ABX was utilized to establish a pseudo-germfree mouse model.C57BL/6J mice were classified into four groups according to simple randomization:HFD/STZ group,HFD/STZ+ABX group,HFD/STZ+EMPA group,and HFD/STZ+ABX+EMPA group,with five mice in each group.All mice were fed with 60%fat high fat diets until euthanasia at the 13th weeks.At the 4th week,the DN model was established by intraperitoneal injection of STZ(50 mg·kg^-1·d^-1)for five consecutive days.At the 6th week,mice were fasted for12 h at night the day before and fasting blood glucose（FBG）was measured.From the8th week,HFD/STZ+ABX and HFD/STZ+ABX+EMPA groups were given ABX（metronidazole 0.5 mg/m L,polymyxin B 1 mg/m L,vancomycin 0.25 mg/m L,ampicillin 0.5 mg/m L）gavage daily,and HFD/STZ and HFD/STZ+EMPA groups were given equal volumes of sterile water gavage daily,for five consecutive weeks.From the 9th week,HFD/STZ+EMPA and HFD/STZ+ABX+EMPA groups were given EMPA（10 mg/kg）gavage daily,HFD/STZ and HFD/STZ+ABX groups were given equal volumes of sterile water gavage daily,for four consecutive weeks.Two days before euthanasia,fresh feces were collected for two consecutive days to detect SCFAs by GC-MS.One day before euthanasia,urine was collected for detection of ACR.After euthanasia,kidney and colon tissues were collected to observe the pathomorphological changes by renal PAS staining and Masson staining,and colon AB-PAS staining and immunofluorescence staining.Results:1.The DN model was successfully constructed.At the 6th week,FBG levels of HFD/STZ group and HFD/STZ+EMPA group were more than 11.1 mmol/L.At the13th week,compared to NFD group,body weight was significantly decreased（P<0.01）,the blood glucose at different times and area under curve（AUC）within 120minutes of OGTT were significantly increased,respectively（P<0.01,P<0.0001）,and urinary albumin creatinine ratio（UACR）was significantly enhanced（P<0.0001）,PAS staining and Masson staining showed diabetic renal pathological changes,destruction of structure of glomeruli and tubules,and glycogen deposition and fibrosis in the HFD/STZ group.2.Empagliflozin ameliorated DN.At the 13th week,compared with HFD/STZ group,body weight was significantly increased（P<0.01）,the blood glucose at different times and AUC within 120 minutes of OGTT were significantly decreased,respectively（P<0.01,P<0.0001）,UACR was significantly reduced（P<0.001）,the structure of glomeruli and tubules roughly restored to normal,and renal glycogen deposition and fibrosis were markedly lessened in the HFD/STZ+EMPA group.3.EMPA regulated gut microbiota and metabolism.EMPA enhanced the gut microbiota diversity,augmented SCFAs-producing bacteria,Bacteroides and Odoribacter,and inhibited LPS-producing bacteria,Oscillibacter.Tax4Fun functional prediction analysis showed that the gut microbiota of mice in the HFD/STZ group was mainly enriched in secretion system,ABC transporters,methane metabolism,bacterial motility proteins,two component system,and quorum sensing.The gut microbiota of mice in the HFD/STZ+EMPA group was mainly enriched in purine metabolism,messenger RNA biogenesis,alanine,aspartate and glutamate metabolism,exosome,amino acid related enzymes,mitochondrial biosynthesis,and pathways such as secretion system,ABC transporters,two component system,and quorum sensing were down-regulated.EMPA significantly increased fecal total SCFAs,acetic acid,butyric acid,respectively（P<0.01,P<0.01,P<0.05）,propionic acid,valeric acid,isobutyric acid and isovaleric acid showed an increasing trend,respectively（P>0.05,P>0.05,P>0.05,P>0.05）,and EMPA significantly decreased fecal LPS（P<0.0001）.4.EMPA alleviated the damage of intestinal mucosal barrier.Compared to HFD/STZ group,the structure of colonic crypt roughly restored to normal,the number of goblet cells and the expressions of Zonula occludens-1（ZO-1）protein and Occludin protein were significantly increased,respectively（P<0.001,P<0.001,P<0.01）,and the serum LPS was significantly decreased（P<0.01）in the HFD/STZ+EMPA group.5.There were some correlations among gut microbiota,SCFAs,LPS,and UACR.Bacteroides was positively related to total SCFAs and acetic acid,respectively（P<0.05,P<0.05）.Odoribacter was positively associated with total SCFAs and butyric acid,respectively（P<0.05,P<0.05）.Oscillibacter was positively related to fecal LPS and serum LPS,respectively（P<0.01,P<0.001）.UACR was positively associated with fecal LPS,serum LPS and Oscillibacter,respectively（P<0.001,P<0.001,P<0.01）.UACR was negatively connected with total SCFAs,acetic acid and butyric acid,respectively（P<0.001,P<0.001,P<0.001）.6.In the pseudo-germfree DN model mice,the effects of EMPA on reducing UACR and renal glycogen deposition and fibrosis were lessened.Compared with the HFD/STZ+ABX+EMPA group,UACR was significantly decreased（P<0.05）,renal glycogen deposition was significantly lessened（P<0.05）,and renal fibrosis showed a decreasing trend（P>0.05）in the HFD/STZ+EMPA group.7.In the pseudo-germfree DN model mice,the effect of EMPA on increasing fecal SCFAs was reduced.Compared with the HFD/STZ+ABX+EMPA group,total SCFAs,acetic acid and butyric acid in feces were significantly increased respectively in the HFD/STZ+EMPA group（P<0.0001,P<0.0001,P<0.001）.8.In the pseudo-germfree DN model mice,the effects of EMPA on increasing the number of goblet cells and the expressions of ZO-1 protein and Occludin protein of colon tissue were lessened.Compared with the HFD/STZ+ABX+EMPA group,the number of goblet cells was markedly increased,the expression of ZO-1 protein was significantly enhanced（P<0.0001）,and the expression of Occludin protein showed an increasing trend（P=0.0618）in the HFD/STZ+EMPA group.Conclusions:1.EMPA alleviated the body weight loss and impaired glucose tolerance,improved kidney function,alleviated the damage of structure of glomeruli and tubules,reduced the renal glycogen deposition and fibrosis in DN model mice,and played the roles in ameliorating metabolic disorder,kidney function and pathological damage,thereby improved DN.2.EMPA regulated gut microbiota and metabolism of DN model mice,increased SCFAs-producing bacteria,Bacteroides and Odoribacter,reduced LPS-producing bacteria,Oscillibacter,enhanced fecal SCFAs,and decreased fecal LPS.3.EMPA ameliorated the injury of the structure of colonic crypt,increased the number of goblet cells and the expressions of ZO-1 protein and Occludin protein,repaired the intestinal mucosal barrier injury,and reduced serum LPS in DN model mice.4.In the state of gut microbiota exhaustion,the effects of EMPA on improving kidney function,reducing renal glycogen deposition and fibrosis were weakened,and failed to play an effective role in ameliorating kidney function and pathological damage.5.In the state of gut microbiota exhaustion,the effect of EMPA on increasing fecal SCFAs was lessened,and failed to play an effective role in regulating gut microbiota metabolism.6.In the state of gut microbiota exhaustion,the effects of EMPA on enhancing the number of goblet cells and the expressions of ZO-1 protein and Occludin protein were weakened,and failed to play an effective role in repairing the damage of intestinal mucosal barrier.To sum up,by regulating gut microbiota,EMPA increased SCFAs-producing bacteria,Bacteroides and Odoribacter,reduced LPS-producing bacteria,Oscillibacter,enhanced fecal SCFAs,decreased fecal LPS,repaired the intestinal mucosal barrier injury,reduced serum LPS,and thus played a role in ameliorating DN.