Microarray microRNA profiling of urinary exosomes in a 5XFAD mouse model of Alzheimer’s disease

Background:Alzheimer's disease(AD)is an incurable and irreversible neurodegen-erative disease,without a clear pathogenesis.Therefore,identification of candidates before amyloid-βplaque(Aβ)deposition proceeds is of major significance for earlier intervention in AD.Methods:To explore the potential noninvasive earlier biomarkers of AD in a 5XFAD mouse model,microRNAs(miRNAs)from urinary exosomes in 1-month-old pre-Aβaccumulation 5XFAD mice models and their littermate controls were profiled by mi-croarray analysis.The differentially expressed miRNAs were further analyzed via droplet digital PCR(ddPCR).Results:Microarray analysis demonstrated that 48 differentially expressed miRNAs(18 upregulated and 30 downregulated),of which six miRNAs-miR-196b-5p,miR-339-3p,miR-34a-5p,miR-376b-3p,miR-677-5p,and miR-721-were predicted to display gene targets and important signaling pathways closely associated with AD pathogenesis and verified by ddPCR.Conclusions:Urinary exosomal miRNAs showing differences in expression prior to Aβ-plaque deposition were identified.These exosomal miRNAs represent potential noninvasive biomarkers that may be used to prevent AD in clinical applications.

Reference gene panel for urinary exosome-based molecular diagnostics in patients with kidney disease

BACKGROUND Kidney disease is a severe complication of diabetes that often leads to end-stage renal disease.Early diagnosis is crucial for prevention or delay.However,the current diagnostic methods,with their limitations in detecting the disease in its early stages,underscore the urgency and importance of finding new solutions.miRNAs encapsulated inside urinary exosomes(UEs)have potential as early biomarkers for kidney diseases.The need for reference miRNAs for accurate interpretation currently limits their translational potential.AIM To identify consistently expressing reference miRNAs from UEs of controls and patients with type 2 diabetesmellitus(T2DM)and biopsy-confirmed kidney diseases.METHODS miRNA profiling was performed on UEs from 31 human urine samples using a rigorous and unbiased method.The UEs were isolated from urine samples collected from healthy individuals(n=6),patients with T2DM(n=13),and T2DM patients who also had kidney diseases(including diabetic nephropathy,n=5;membranous nephropathy,n=5;and IgA nephropathy,n=2)through differential ultracentrifugation.After characterizing the UEs,miRNA expression profiling using microarray technology was conducted.RESULTS Microarray data analysis identified 14 miRNAs that were consistently expressed in UEs from 31 human samples,representing various kidney conditions:diabetic controls,diabetic nephropathy,membrane nephropathy,IgA nephropathy,and healthy controls.Through in silico analysis,we determined that 10 of these miRNAs had significant potential to serve as reference genes in UEs.CONCLUSION We identified uniformly expressing UE miRNAs that could serve as reference genes kidney disease biomarkers.

Urinary exosomal microRNA-145-5p and microRNA-27a-3p act as noninvasive diagnostic biomarkers for diabetic kidney disease

BACKGROUND Diabetic kidney disease(DKD),characterized by increased urinary microalbumin levels and decreased renal function,is the primary cause of end-stage renal di-sease.Its pathological mechanisms are complicated and multifactorial;Therefore,sensitive and specific biomarkers are needed.Urinary exosome originate from diverse renal cells in nephron segments and partially mirror the pathological changes in the kidney.The microRNAs(miRNAs)in urinary exosome are remark-ably stable and highly tissue-specific for the kidney.METHODS Type 2 diabetic mellitus(T2DM)patients were recruited from the Second Hospital of Hebei Medical University and were divided into two groups:DM,diabetic pa-tients without albuminuria[urinary albumin to creatinine ratio(UACR)<30 mg/g]and DKD,diabetic patients with albuminuria(UACR≥30 mg/g).Healthy subjects were the normal control(NC)group.Urinary exosomal miR-145-5p,miR-27a-3p,and miR-29c-3p,were detected using real-time quantitative polymerase chain reaction.The correlation between exosomal miRNAs and the clinical in-dexes was evaluated.The diagnostic values of exosomal miR-145-5p and miR-27a-3p in DKD were determined using receiver operating characteristic(ROC)analysis.Biological functions of miR-145-5p were investigated by performing RESULTS Urinary exosomal expression of miR-145-5p and miR-27a-3p was more upregulated in the DKD group than in the DM group(miR-145-5p:4.54±1.45 vs 1.95±0.93,P<0.001;miR-27a-3p:2.33±0.79 vs 1.71±0.76,P<0.05)and the NC group(miR-145-5p:4.54±1.45 vs 1.55±0.83,P<0.001;miR-27a-3p:2.33±0.79 vs 1.10±0.51,P<0.001).The exosomal miR-145-5p and miR-27a-3p positively correlated with albuminuria and serum creatinine and negatively correlated with the estimated glomerular filtration rate.miR-27a-3p was also closely related to blood glucose,gly-cosylated hemoglobin A1c,and low-density lipoprotein cholesterol.ROC analysis revealed that miR-145-5p had a better area under the curve of 0.88[95%confidence interval(CI):0.784-0.985,P<0.0001]in diagnosing DKD than miR-27a-3p with 0.71(95%CI:0.547-0.871,P=0.0239).Bioinformatics analysis revealed that the target genes of miR-145-5p were located in the actin filament,cytoskeleton,and extracellular exosome and were involved in the pathological processes of DKD,including apoptosis,inflammation,and fibrosis.CONCLUSION Urinary exosomal miR-145-5p and miR-27a-3p may serve as novel noninvasive diagnostic biomarkers or promising therapeutic targets for DKD.

In-depth urinary and exosome proteome profiling analysis identifies novel biomarkers for diabetic kidney disease

Diabetic kidney disease(DKD)is a major microvascular complication of type 2 diabetes mellitus(T2DM).Monitoring the early diagnostic period and disease progression plays a crucial role in treating DKD.In this study,to comprehensively elucidate the molecular characteristics of urinary proteins and urinary exosome proteins in type 2 DKD,we performed large-scale urinary proteomics(n=144)and urinary exosome proteomics(n=44)analyses on T2DM patients with albuminuria in varying degrees.The dynamics analysis of the urinary and exosome proteomes in our study provides a valuable resource for discovering potential urinary biomarkers in patients with DKD.A series of potential biomarkers,such as SERPINA1 and transferrin(TF),were detected and validated to be used for DKD diagnosis or disease monitoring.The results of our study comprehensively elucidated the changes in the urinary proteome and revealed several potential biomarkers reflecting the progression of DKD,which provide a reference for DKD biomarker screening.