Screening And Application Of Aristolochic Acid Degrading Bacteria And Molecular Markers Of Nephrotoxicity

ObjectivesThe objectives of this study are as follows:(1)to screen strains which degrade aristolochic acid I,and then select the most efficient degrader for further analysis and identification;(2)to explore the cellular effect and mechanism of kidney injury caused by aristolochic acid I,and screen biomarkers for detecting its nephrotoxicity;(3)to apply the degrader to ferment Qingmuxiang which contains high level of aristolochic acid I,and then evaluate nephrotoxicity using the biomarkers.MethodsFirst,strains which capable of degrading AAI were isolated from soil samples collected from rhizosphere of Aristolochia debilis.Second,after comparing the degradation ability of these strains,the most efficient degrader was selected for further identification,and the whole genome sequence of it was determined for comparative genomic analysis.Third,sequencing data of renal cell carcinoma and AAI induced kidney injury were collected from TCGA and NCBI,and then enrichment analysis and signal pathway analysis were carried out to explore the molecular mechanism of renal injury induced by AAI.Next,potential biomarkers for detecting AAI nephrotoxicity were screened and validated.Finally,the medicinal materials containing high level of AAI were fermented by the strain,and the nephrotoxicity was evaluated using biomarkers screened in this study.ResultsThe main results are as follows:(1)Thirty-three strains were isolated from soil samples collected from rhizosphere of Aristolochia debilis,11 of which had different AAI degradation ability,and the highest degrading rate was 85.36% in 48 hours under non-optimal conditions.Six strains without AAI degradation ability were isolated from rhizosphere soil of camphor trees.(2)The 16 S r RNA gene sequence of the most efficient degrader was sequenced.Based on the 16 S r RNA sequence,the strain was identified as a potential new species of the Sphingobium genus,tentatively named Sphingobium sp.AA3.(3)The second generation of 500× genome data of Sphingobium sp.AA3 was obtained with good quality,and 78 contigs were assembled with a total genome size of 5,538,228 bps.The genome of the strain differs greatly from its close relatives,which supports that the strain is a new species of the Sphingobium genus.The whole genome contains 5,663 genes,of which 5,581 are coding genes,and 73% of coding genes have been functionally annotated.Nearly one third of the annotated genes are metabolic related proteins,and many of them may be related to aromatic compounds degradation.(4)Changes of gene expression induced by AAI were similar to primary renal cell carcinoma in metabolic pathway,and exhibited Warburg effect both;in addition,AAI and primary RCC both up-regulate PI3 K pathway,but through different PI3 K pathway proteins.AAI also showed simultaneously up-regulated cell apoptosis and necrosis pathways;multiple genes showed the same expression pattern in aristolochic acid nephropathy and primary renal cell carcinoma,some of which were closely related to the survival and prognosis of RCC patients.(5)Animal experiments showed that Acad11 and Enpep were down-regulated while PANX2 and WNT7 B were up-regulated in AAI induced renal injury,which were consistent with the data of primary renal cancer.Meanwhile,the down-regulation of Prima1 and the up-regulation of Cdkn1 a were the potential molecular markers of renal cancer induced by AAI,for which were different from the pathogenesis of primary renal cancer.(6)Sphingobium sp.AA3 showed effective AAI-degradation ability in Qingmuxiang decoction,with a degradation rate of more than 90%,detecting by HPLC method.(7)Animal experiments showed that Qingmuxiang decoction and AAI standard solution could induce significant changes in the kidney shape and the expression of Prima1,Wnt7 b and Cdkn1 a in mice,while the results of Sphingobium sp.AA3 fermented Qingmuxiang decoction were almost the same as those in saline control group,suggesting that the use of Sphingobium sp.AA3 fermentation may effectively reduce acute kidney toxicity of large dose of Qingmuxiang decoction.ConclusionsEleven AAI-degrading strains were isolated from rhizosphere soil of Aristolochia debilis,and their degradation ability varied,with strain AA3 the highest.Based on the 16 S r RNA sequence,strain AA3 was identified as a potential new species of the Sphingobium genus.Its whole genome sequence was determined by the second generation sequencing technology,and analyzed.Thirteen potential biomarkers of AAI nephrotoxicity,including Prima1,Acad11,Enpep,Epha2,Psmb8,Bcl3,Axl,Hgfac,Cd276,Mmp2,PANX2,WNT7 B and Cdkn1 a,were screened by serial analysis and validated by q-PCR.Sphingobium sp.AA3 was used to ferment Qingmuxiang decoction,and degraded AAI efficiently of the decoction.