Abstract: TH-OR132

Transcriptome of Urinary Extracellular Vesicles in Diabetic Kidney Disease

Session Information

Category: Diabetes

  • 501 Diabetes Mellitus and Obesity: Basic - Experimental

Authors

  • Barreiro, Karina A, Institute for Molecular Medicine Finland FIMM, University of Helsinki,, Helsinki, Finland
  • Dwivedi, Om, University of Helsinki, Helsinki, Finland
  • Puhka, Maija, Institute for Molecular Medicine Finland FIMM, Helsinki, Finland
  • Forsblom, Carol, Helsinki University Central Hospital, Helsinki, Finland
  • Groop, Per-Henrik, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
  • Groop, Leif, Lund University, Maimo, Sweden
  • Huber, Tobias B., University Medical Center Hamburg, Hamburg, Germany
  • Holthofer, Harry B., University of Helsinki, Helsinki, Finland
Background

We compared methods for the urinary extracellular vesicle (uEV) harvest and respective transcriptomes in diabetic kidney disease (DKD). This also included study of storage conditions , in -20°C vs. -80°C, to find out whether existing large sample collections stored at -20°C can be successfully used.

Methods

Patients: Type 1 diabetic patients (T1D) and normoalbuminuric controls were included.
Main Measurables: EVs were isolated from 40ml of 24h urines by differential centrifugation, Hydrostatic Filtration Dialysis (HFD) or kit-based isolation. Quality of the EV yield was analyzed with EM and Western blotting. Isolated RNAs were profiled with Bioanalyzer Pico kit and subjected to RNAseq after cDNA library preparation using ultra-low amount protocols. RNAseq was performed using HiSeq 2000 (Illumina) pair-end (2X100) protocol. Output reads were aligned to human reference genome and counted using GENCODE gene annotations. We used gene length normalized values FKPM (Fragments Per Kilobase Of Exon Per Million) as expression measurement for genes.

Results

Results: The isolated EVs appeared typical at EM. All samples had >15 million RNA eads. On average, expression (FKPM >1) of 13,161 genes and a high expression (FPKM≥5) of key kidney specific genes (e.g. SLC12A3, SLC12A1, LGALS1, ATP6V1B1, NPHS2, AQP3, AQP2, SLC22A12). A comprehensive analysis of 182 kidney specific genes revealed >70% (total 132) of the genes in urine EVs. Principal component analysis of these discriminated macro-albuminuric from normoalbuminuric T1D patients. Six genes were differentially expressed in DKD (Puncorrected <0.001 and fold change >1.5 or <0.66). The highest expressed genes were enriched (P>10-11) in pathways of cellular metabolism (oxidative phosphorylation and TCA cycle), mitochondrial, ribosomal and vesicle trafficking functions. Pathway and gene enrichment analyses implicate (P<0.002) TGF-beta signaling , PI3K-Akt signaling and immune pathway in DKD.

Conclusion

uEV transcriptome captures a significant kidney specific transcriptome which differentiates macroalbuminuric from normoalbuminuric T1D patients. Technically, samples stored at different temperatures cannot be directly compared with each other.
Our results show high value of urinary EV transcriptome for biomarker search and call for meticulous standardization of protocols used.