The Study Of Untargeted Urine Metabolomics Of Patients With Diabetic Kidney Disease

ObjectiveTo understand the differences in laboratory-related renal function indexes of patients with diabetic kidney disease(DKD)with different levels of albuminuria,and to analyze the characteristics of urine metabolite profiles with DKD patients,and to screen for the potential biomarkers related to DKD,to provide new ideas for the study of biomarkers such as the prediction,diagnosis and detection of kidney damage in DKD patients.MethodsA total of 43 diabetes mellitus patients and 64 DKD patients who were treated in Mianyang Central Hospital from March 2019 to December 2019 as the research subjects.And DKD patients were divided into albuminuria DKD(ADKD)group and normoalbuminuric DKD(NADKD)group based on UACR levels.Urine and blood samples were collected from all subjects,detecting serum creatinine(SCr),serum cystatin C(SCys C),Urea,complement C1 q subunit(C1q),neutrophil gelatinase-associated apolipoprotein(NGAL)and uric acid(UA),urine creatinine(UCr)and urine albumin(UAlb)levels of all subjects,e GFR and UACR were calculated based on SCys C,UAlb and UCr.All subjects were analyzed for urine metabolites using non-targeted metabolomics based on UPLC-MS/MS,and multivariate statistical analysis,nonparametric testing,and metabolic pathways analysis were used to analyze the differential urine metabolites between each group,then,correlation analysis and ROC were used to screen differential metabolites related to DKD.Results1.The results of the analysis of the difference between the observed indicators showed that the SDM group,NADKD group and ADKD group had significant differences in age and the level of UAlb,UACR,Urea,SCr,UA,SCys C,e GFR and NGAL(all P<0.05).Compared with SDM patients,NADKD patients was older,and the level of SCr,SCys C and UA were increased significantly(all P<0.05),while the level of e GFR was reduced significantly(all P<0.05).ADKD patients was older,and the level of UAlb,UACR,SCr,SCys C,UA and NGAL were increased significantly compared with SDM patients(all P<0.05),while the level of e GFR was reduced significantly(all P<0.05);However,compared with the NADKD group,only the levels of UAlb and UACR were increased significantly in the ADKD group(all P<0.05),and there were no significant differences in other laboratory kidney function indicators(all P>0.05).The results of the ROC analysis of observed laboratory indicators showed that when diagnose NADKD or ADKD patients from SDM patients,the diagnostic performance of SCr was the highest(AUC is 0.833 and 0.702 respectively),when diagnose DKD(NADKD+ADKD)patients from SDM patients,the diagnostic performance of SCr(AUC=0.768)was the highest.2.UPLC-MS/MS was used to analyze the urine metabolite profiles of the all subjects.It was showed that the urine metabolite profiles of the three groups were different.Through further analysis,53 different metabolites were screened between SDM group and NADKD group,84 different metabolites were screened between SDM group and ADKD group,and 48 different metabolites were screened between NADKD group and ADKD group,79 different metabolites were screened between the SDM group and the DKD group.Classifying these metabolites,it was found that the metabolites that accounted for the most among the three groups were mainly dispersed in two major substances:(1)lipids and lipid-like molecules,(2)amino acids,peptides,and analogues.In addition,compared with the SDM group,the levels of various lipid-related metabolites(including dehydroepiandrosterone,1-hexadecanol,γ-linolenic acid,palmitoleic acid,arachidic acid,allocholic acid,oleic acid,cholesterol and alpha-tocotrienol)were increased significantly in ADKD group(all P<0.05),however,NADKD group had no significant changes in these lipid-related metabolites(all P>0.05).3.The results of metabolic pathway analysis showed that compared with the SDM group,the metabolic pathways related to DKD mainly involved the alanine,aspartic acid and glutamate metabolism,the citric acid cycle,the cysteine and methionine metabolism,the arginine and proline metabolism,and the tyrosine metabolism;the metabolic pathways related to NADKD mainly involved the β-alanine metabolism,the citric acid cycle,the pyrimidine metabolism,the alanine,aspartic acid and glutamate metabolism;the metabolic pathways related to ADKD mainly involved the citric acid cycle,the alanine,aspartic acid and glutamate metabolism.The metabolic pathway that significantly changed between the NADKD group and the ADKD group was β-alanine metabolism.4.Using the analysis of ROC to screen out 14 kinds of urine metabolites that can be used to diagnose DKD,including dehydroepiandrosterone,1-hexadecanol,α-tocopherol,γ-linolenic acid,acetyl phosphate,d AMP,mannitol,L-erythro-4-hydroxyglutamic acid,N-carbamoyl putrescine,uridine,5-aminovaleric acid,(S)-5-diphosphomevalonic acid,all-trans retinoic acid and cis-aconitic acid.Meanwhile,5-aminovaleric acid,Ncarbamoyl putrescine,γ-linolenic acid,all-trans retinoic acid,and α-tocopherol were correlated with UACR and e GFR.Conclusions1.The changes of SCr,UA,SCys C and e GFR in the NADKD group and the ADKD group were consistent,and it was showed that the kidney function of NADKD patients still had varying degrees of damage,although there was no increase in urine albumin with NADKD patients,and ROC analysis found that the observed renal function indicators were not good for the diagnosis of DKD.2.Urine metabolomics analysis showed that there were differences among the SDM group,NADKD group and ADKD group significantly.It was suggested that urine metabolomics analysis technology could be used in research of ADKD and NADKD laboratory diagnosis and pathogenesis.3.Changes in the levels of lipids and lipid-like molecules,as well as amino acids,peptides and analogues might be closely related to DKD.In addition,the change of the alanine,aspartic acid and glutamate metabolism,as well as the citric acid cycle was closely related to the occurrence of DKD.The cysteine and methionine metabolism,the arginine and proline metabolism,and the tyrosine metabolism might be related to DKD.And the β-alanine metabolism was closely related to the occurrence of NADKD.4.Urine metabolites of 5-aminovaleric acid,N-carbamoyl putrescine,γ-linolenic acid,all-trans retinoic acid,and α-tocopherol were both correlated with UACR and e GFR,and had suggested that it had the potential to diagnose DKD.