Poria Cocos Ameliorates Chronic Kidney Disease And Its Mechanism By Microbiome-metabolomics

Chronic kidney disease(CKD)is one of the chronic diseases that poses a great threat to human health,and has become a public health issue of great concern.Poria cocos,a traditional Chinese medicinal,is used to treat edema and insomnia,which is now commonly used to treat CKD clinically.Studies have shown that gut dysbiosis exacerbates kidney damage.Gut microbiota as the main source of uremic toxins is associated with CKD and metabolites produced by gut microbiota enter the blood circulation through the disrupted intestinal epithelial barrier,stimulating cells to produce local or systemic inflammation.Consequently,this study investigated the effect of Poria cocos on intestinal flora and metabolite disorders.Objective:The differential gut microbiome and serum metabolites in 5/6 nephrectomized(NX)-induced CKD rats were identified,and the relationship between gut microbiota and serum metabolites in CKD were explored,based on 16 S r RNA gene sequencing technology and non-targeted metabolomics.To study the effects of ethanol extract of poria cocos(PC)and its main component poricoic acid A(PAA)on the identified microbiota-related metabolites,and intestinal epithelial barrier,inflammation and oxidative stress.This study provides reference theory and scientific basis for revealing the effects and mechanisms of Poria cocos on renal fibrosis,and provides scientific reference for clinical treatment of CKD with Poria cocos.Methods:1.Alterations of gut microbiota structure and composition in CKD rats.The CKD rat model induced by NX was used to collect 24-hour urine,blood and intestinal contents of the rats in each group,and the physiological and biochemical indicators such as blood pressure,urine protein and serum creatinine of the rats in each group were determined.16 S r RNA sequencing was carried out on the samples of colon contents to obtain the data of intestinal microbiome.The changes of intestinal flora in CKD rats were studied based on the analysis methods of diversity analysis,species composition analysis and correlation analysis.The potential functional impact of altered gut microbiota in CKD rats was investigated by PICRUSt metabolic function prediction analysis.2.Correlation analysis of microbiome and serum metabolomics in CKD rats.Differential metabolites in serum of CKD rats were identified based on UPLC-HDMS.Correlation analysis was used to explore the relationship between differential metabolites related to intestinal flora imbalance and systolic blood pressure(SBP)and creatinine clearance(CCr),and to determine the relevant metabolic pathways.3.Improvement effect of PAA and PC intervention on CKD rats.PAA and PC were used to intervene in CKD rats,and the metabolites of related metabolic pathways were analyzed by targeted metabolomics technology,and the effects of PAA and PC on the metabolite disturbances of related metabolic pathways were studied.Western blot was used to analyze the expression of tight junction proteins such as ZO1 and Occludin in colon tissue of rats in each group,and the expression of proteins related to pro-inflammatory IκBα/NF-κB signaling pathway and anti-inflammatory Keap1/Nrf2 signaling pathway in kidney tissue.Through histopathological analysis(PAS,Masson’s trichrome and immunohistochemical staining),the effects of PAA and PC on kidney injury and the expression of α-SMA,Vimentin and Collagen I fibrogenic factors in kidney tissues of rats in each group were investigated.Results:1.Alteration of colonic microbiota in the NX rats.The microbial communities were significantly different at 13 genus levels in NX rats,such as Allobaculum,Blautia,Pseudomonas and Clostridium＿IV,compared with the sham rats.These 13 genera were related to the changes of physiological and biochemical indicators,including SBP,CCr and creatinine though cytoscape software analysis.The altered gut microbiota was associated with 33 metabolic pathways including amino acid metabolism,nucleotide metabolism,and lipid metabolism.The significant positive correlations between increased proteinuria with Blautia and decreased hematocrit with Clostridium＿IV.PICRUSt analysis showed that altered gut microbiota may have an impact on 33 metabolic pathways.2.Alteration of serum metabolome in NX rats.291 differential metabolites including,glycine,spermine and spermidine were identified by UPLC-HDMS.These metabolites are mainly involved in fatty acid metabolism,amino acid metabolism,polyamine metabolism and purine metabolism.3.Association of the NX-induced gut microbial dysbiosis with dysregulation metabolites.36 metabolites,including glycine,spermine and spermidine were significantly associated with 13 altered microbial genera,including Allobaculum,Blautia,Pseudomonas and Clostridium＿IV.16 metabolites were significantly associated with SBP,among which 10 metabolites including glycine,hippuric acid and phenylalanine were associated with glycine metabolism.15 metabolites were significantly associated with CCr,among which 7 metabolites including putrescine,spermine and spermidine were polyamine metabolites.Based on the correlation network analysis of 9 clinical indicators,13 genus-level bacteria,7 glycine metabolites and 6 polyamine metabolites,it was glycine metabolism was correlated with intestinal flora imbalance and SBP,polyamine metabolism was correlated with intestinal flora imbalance and CCr in NX rats.4.PAA and PC can improve the disturbance of related metabolites of dysregulated intestinal flora in NX rats.PAA and PC ameliorated the serum abnormal glycine and polyamine metabolism,alleviated the disruption of gut barrier,regulated pro-inflammatory IκBα/NF-κB signaling pathway and anti-inflammatory Keap1/Nrf2 signaling pathway to inhibit inflammation and oxidative stress in NX rats.Histological analysis indicated that both PAA and PC significantly attenuated interstitial inflammatory cell infiltration,tubular atrophy/dilatation and the upregulation of profibrotic factors.Compared with PC,PAA exhibited stronger activity against renal fibrosis.Conclusion:This study showd that NX led to the dysbacteriosis and disturbance of serum glycine in the colon of rats,and the disordered intestinal flora and metabolites are related to glycine and polyamine metabolites.PAA and PC mitigated microbial dysbiosis,attenuated oxidative stress,inflammation and renal fibrosis,and retarded the decline of renal function in NX rats.