Ketoacids Analogues Delaying The Progression Of CKD By Regulating Intestinal Flora And Changing Substance Metabolism

Objective: The high prevalence and rising incidence of chronic kidney disease(CKD)pose a huge economic burden.Ketoacids can supplement the essential amino acid of the body without increasing the burden on the kidneys and can delay the progression of CKD,but the mechanism is not completely clear.There is a complex interaction between the intestinal flora and the kidney,and CKD interferes with the symbiotic relationship between the host organ and the intestinal microflora.Therefore,this study intends to explore the effects of KA on the intestinal flora and serum metabolites of CKD mice,provide a theoretical reference and scientific basis for the clinical use of KA in the treatment of CKD,and further explore the intestinal flora and metabolic characteristics of CKD.Methods: 1.Establishment of CKD mouse model: C57 mice were selected and divided into control group,LPD group and KA group.Unilateral ureteral obstruction(UUO)model was selected,after 28 days of intervention,stool,blood and kidney samples were collected to determine serum creatinine and blood urea nitrogen.2.Intestinal microbiota analysis: 16 s RNA sequencing was sequenced from stool samples,and the changes of intestinal flora in CKD micewere studied by diversity analysis,species composition analysis,correlation analysis.3.Serum metabolomics research: Differential metabolites in mice serum were identified based on ultra-high performance liquid chromatography-high-definition mass spectrometry(LC-MS)technology.The metabolic pathway was subsequently enriched,and metabolic pathway was mainly involved in the screening of differential metabolites.4.Correlation between intestinal microbiota and differential metabolites: Pearson correlation analysis was used to explore the relationship between the change of intestinal microflora and differential metabolites in CKD mice.Results: 1.Biochemical indexes: In control group,serum creatinine was21.08 ± 5.83 μmol/L,blood urea nitrogen was 3.75 ± 1.89 mmol/l.In LPD group,serum creatinine was 85.73 ± 14.21 μmol/L,blood urea nitrogen was19.46 ± 3.36 mmol/l.In KA Group,serum creatinine was 56.93 ± 8.86 μmol/L,blood urea nitrogen was 16.70 ± 5.58 mmol/L.The serum creatinine and blood urea nitrogen were significantly increased in KA and LPD groups(F=60.03,p<0.001;F=27.48,p<0.001).2.Changes in intestinal microbiota of CKD mice: α diversity analysis showed that the Shannon indices of the control group,KA group and LPD group were 5.970±0.91,6.42±0.48,5.32±0.41,with no statistically significant difference(F =3.76,p=0.05).Simpson’s index was 0.92±0.08,0.97±0.01,0.91±0.04,respectively,with no significant difference(F=0.67,p=0.527).At the phylum level,the Firmicutes(p=0.014,p=0.007)and Desulfurization(p=0.04,p=0.01)were significantly increased in both KA and LPD groups.At the genus level,compared with the control,the levels of Vibrio desulfurizome(p=0.001)and Escherichia coli(p=0.003)in the KA group were significantly increased,while the levels of Lactobacillus(p=0.039),Prevotella(p=0.015)and Clostridium difficile(p=0.005)were significantly decreased.Compared with the LPD group,the level of Alistipes(p=0.021)was significantly increased,Odoribacter(p=0.003)was significantly decreased in the KA group.3.Changes in serum metabolites of CKD mice: Compared with the LPD group,citric acid,citrulline,L-acetyl-carnitine,L-(+)-gluconose,galactose,glutathione,Ganglioside,dimethylarginine,Glutathione,propionylcarnitine,L-palmitoylcarnitine,Lyso PC(20:4(8Z,11 Z,14Z,17Z)),3-(3-INDOLYL)-2-oxopropionic acid were significantly increased in KA Group,serine phosphate,antipyrine,PFC Pirin Blagoevgrad and xanthine decreased significantly.These metabolites are mainly concentrated in the metabolism of choline,Fatty acid metabolism(linoleic acid,Glycerophospholipid),amino acid metabolism(arginine biosynthesis,alanine,L-Aspartic Acid and glutamate metabolism)in cancer,tricarboxylic acid cycle(TCA cycle),GABA-synapses and other metabolic pathways.4.The relationship between changes of intestinal flora and differential metabolites in CKD mice: Alistipes was positively correlated with citrulline and PC(14:0/20:2(11Z,14Z))(r=-0.49,p=0.041).The intestinal group of Wenken-RC9 was positively correlated with serine phosphate level(r=0.53,p=0.026),and it was negatively correlated with citrulline(r =-0.49,p = 0.041),glutarate(r =-0.64,p = 0.006),glucose(r =-0.49,p = 0.041)and Glutathione(r =-0.67,p = 0.003).Helicobacter pylori was negatively associated with citric acid(r=-0.49,p=0.041)and citrulline(r=-0.49,p=0.041).Conclusion: 1.UUO model can be used as an animal model for CKD research.2.In CKD mice,firmicutes and desulphurization bacteria increased significantly,Lactobacillus and prevotella decreased,and Escherichia coli increased.3.KA can affect the intestinal flora of CKD mice,change the level of serum metabolites,and delay the progression of CKD.4.KA may affect arginine biosynthesis by up-regulating citrulline.5.Ka may affect alanine,L-Aspartic Acid and glutamate metabolism by up-regulating citric acid.