Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Capturing the Kidney Transcriptome by Urinary Extracellular Vesicles-From Pre-Analytical Obstacles to Biomarker Research.

Author(s): Barreiro K, Dwivedi OP, Rannikko A, Holthöfer H, Tuomi T, Groop PH, Puhka M

Publication: Genes (Basel), 2023, Vol. 14, Page

PubMed ID: 37510317 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the miRNA and mRNA profile of uEVs isolated from matched urine stored at -20°C or -80°C for 13-16 months, in uEVs isolated using different methods, and uEVs isolated from patients with diabetic kidney disease (DKD) and healthy volunteers. The authors also identified stable mRNAs for potential use as a reference. This study mined mRNA and miRNA sequencing data of uEVs. Unless otherwise specified, uEVs were isolated by ultracentrifugation on the day of collection. To investigate the effects of frozen storage temperature, miRNA and mRNA levels were compared in 24 h urine specimens collected from four men with type I diabetes (two with macroalbuminuria); urine specimens were immediately aliquoted and stored at -20°C or -80°C for 13-16 months before uEV isolation.  To investigate the effects of isolation method, uEVs were isolated from the urine of 5 healthy men and 5 male patients with type 1 diabetes using ultracentrifugation, hydrostatic filtration dialysis, and the Norgen Urine Exosome Purification and RNA Isolation Midi Kit. To identify miRNAs associated with diabetic kidney disease (DKD), levels of 170 miRNAs suspected to be involved in DKD were compared in ultracentrifugation-isolated uEVs from 72 patients with DKD (38 normoalbuminuric, 15 microalbuminuric, and 19 macroalbuminuric) and healthy volunteers, and a second cohort of 30 patients with DKD (18 normoalbuminuric, 8 microalbuminuric, and 4 macroalbuminuric).   To identify stable mRNA, the 100 least variable mRNA were identified in the isolation method cohort (specimens processed with the Norgen Kit were excluded), the two DKD cohorts, and three additional cohorts (a total of 35 men with Type 1 diabetes and 12 healthy men) and were validated in urine from a prostate cancer cohort (3 prostate cancer patients and one healthy male) and a cohort of 30 women with type 2 diabetes. Unless otherwise specified, uEVs were isolated by ultracentrifugation on the day of collection.

   Summary of Findings:

   Of the 40 most abundant miRNAs and the 56 most abundant mRNAs in kidney, 29 miRNAs and 33 mRNAs were expressed in urine specimens stored at different frozen storage temperatures. Normalized levels of 22 of these miRNAs and 15 of the mRNAs were lower when urine specimens were stored at -20°C than -80°C.  In the differential expression analysis of urine stored for up to 1 year, normalized counts of 29 miRNAs were lower and 4 miRNAs were higher when urine was stored at -20°C than -80°C; of these 33 miRNAs, 25 were previously reported to be associated with kidney disease. In the comparison of EVs isolated using different methods, 36 miRNAs and 31 mRNAs were detected. Compared to EVs isolated by ultracentrifugation, EVs isolated by hydrostatic filtration dialysis had lower levels of 18 miRNAs (3-58% lower), and EVs isolated using the Norgen Kit had lower levels of 21 mRNAs. Finally, in comparing levels of 170 miRNAs suspected to be associated with diabetic kidney disease in uEVs from patients with DKD and healthy controls, 39 were differentially expressed and 31 could successfully separate uEVs from healthy patients from those with DKD. Among the most affected miRNAs, lower levels of miR-30b-5p, miR-221-3p, let-7f-1-3p, let-7a-3p and miR-15b-5p were found in patients with DKD, with the direction of change in miR-15b-5p contrary to literature-based expectations. Levels of miR-424-5p, miR-486-3p, miR-335-5p, and miR-126-3p were all higher in patients with DKD than in healthy controls.  Levels of the 31 DKD-associated miRNAs evaluated did not separate uEVs from prostate cancer patients and healthy volunteers.  A total of 32 mRNAs were identified as potential normalizers based on being among the 100 mRNAs with the lowest CV in all the investigated male cohorts (Norgen workflow and storage temperature cohorts were excluded due to low expression). Investigation of these 32 mRNAs revealed that 18 also had a low CV in the urine from a cohort of 30 women with type 2 diabetes and 11 of these (HSPD1, SRSF3, VAPA, RAB1A, MORF4L1, PGK1, RHOA, UBE2D3, DAZAP2, UBC, and ACTG1) had a low CV in the urine from a cohort of 8 men with prostate cancer. In comparing variability in counts per million, ACTG1 had comparable variability to GAPDH, and like GAPDH, had high counts per million relative to the other mRNAs with low variability.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Diabetes Type 1
   • Normal
   • Neoplastic - Carcinoma
   • Diabetes Type 2

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Diabetic kidney disease Prostate Cancer Healthy
   Analyte Extraction and Purification	Analyte isolation method	Ultracentrifugation Hydrostatic filtration dialysis Norgen Urine Exosome Purification
   Storage	Storage temperature	-20°C -80°C

   View more