Journal: iScience

Article Title: Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease.

doi: 10.1016/j.isci.2023.106686

Figure Lengend Snippet: Figure 1. Study design and Urinary extracellular vesicles (uEV) quality control (A) Study design for the next generation sequencing of uEV mRNAs to assess urine collections, quality and reproducibility (technical comparisons), origins of uEV transcripts (uEV on Tissue map), and discovery of candidate non-invasive markers for diabetic kidney disease (DKD marker candidates). (B–D) Transmission electron micrographs showing uEV of typical morphology and variable sizes; filamentous structures compatible with Tamm-Horsfall protein filaments are visible in the in-set B1. (E) Western blotting showing the presence of uEV-enriched markers CD9 and PDX in uEV preparations. (F) Representative electropherograms of RNA isolated from uEV and analyzed with bioanalyzer using Agilent Pico kit. An RNA peak between 25 and 200 nt was detected in all samples and with this characteristic shape passed quality control. (G) Examples showing signs of nucleic acid degradation (arrows) in uEV RNA samples and not passing quality control. 24 h (24h); albumin excretion rate (AER); blood pressure (BP); body-mass index (BMI); estimated glomerular filtration rate (eGFR); waist-to-hip ratio (WHR); Genotype-Tissue Expression (GTEx) project; glycated hemoglobin (HbA1C); Lymph Node Carcinoma of the prostate (LnCaP), macroalbuminuria (Macro); microalbuminuria (Micro); non-diabetic control (Control); normoalbuminuria (Normo); messenger RNA sequencing (mRNAseq); Podocalyxin (PDX), type 1 or type 2 diabetes (T1D, T2D); urinary extracellular vesicles (uEV); principal component analysis (PCA); single-nucleus RNA sequencing (snRNA-seq).

Article Snippet: DIREVA samples were collected with PI and centrifuged at 1800g for 10 min at +4 C before freezing as above, and FinnDiane samples were collected either with or completely without these steps. iBEAt samples all contained PI (Complete ULTRA, Mini, EDTA-free, Roche; half tablet per 50 ml urine) and were centrifuged at 3000g for 10 min at RT, after which citrate-EDTA buffer was added (1,0 M citrate, 0,1 M EDTA; 2,5 ml per 45 ml urine) before freezing at -80 C. The uEV were isolated from 30 ml of urine (DIREVA and part of FinnDiane) as above except using a SW-32 rotor (129 168 gmax, k-factor 276, Beckmann-Coulter) and from 8 ml of urine (iBEAt and part of FinnDiane) as described (Barreiro et al.23) using a 70.1 ti rotor (82 656 gmax, k-factor 202, Beckman Coulter, Inc., Brea, CA) for 2 h 30 min at 30,000 rpm at +4 C and no wash. uEV protein and particle quality control Urinary EV samples were analyzed as explained previously.22,66 Briefly, Western blotting of uEVmarker proteins was performed using antibodies against CD9 (SC-13118, Santa Cruz) and podocalyxin (clone 3D3, Novus Biologicals).

Techniques: Control, Next-Generation Sequencing, Marker, Transmission Assay, Western Blot, Isolation, Expressing, RNA Sequencing