Figure 2
Identification of tRNAs in extracellular vesicles from urine and validation of tRNAs differentially released in extracellular vesicles from renal ischemia/reperfusion injury (IRI). (a) Immunoblotting analysis showing the indicated proteins in extracellular vesicle (EV) fraction. As a control experiment, resuspended extracellular vesicles were spun at 200 K × g to produce extracellular vesicle (EV)-depleted condition. (b) U6 snoRNA of urinary extracellular vesicles as an adequate reference gene in this experimental condition. Note that the Cq distributions of U6 from urinary extracellular vesicles in IRI were not statistically different. Shown are relative distributions of quantification cycle, Cq of U6 snoRNA from extracellular vesicles of IRI urine normalized to that of sham urine (volume). All urine samples were collected from 24 h after IRI surgery. (c) Among urinary tRNAs with decreased expression 24 h after IRI compared with those from sham, tRNAAla, tRNAGlu, tRNAAsp, and tRNAValAAC, but not tRNAValCAC are mainly present in 200 K × g pellets. The bar graph shows the relative levels of the indicated tRNAs from rat urine either in the pellets or supernatants from 200 K × g ultracentrifugation. n.s. statistically non-significant; *p < 0.05. (d) Relative abundance of urinary tRNAs from renal IRI that are associated with urinary extracellular vesicles fractionation. Expression levels of the indicated tRNA were normalized using U6 snoRNA within each group, and then relative expression values of tRNA of IRI group normalized to those of sham group were plotted. EV, extracellular vesicles. n.s. statistically non-significant; *p < 0.05. (e) Relative abundance of benzonase-resistant, urinary extracellular vesicle tRNAs from renal IRI. Expression levels of the indicated tRNA were normalized using U6 snoRNA within each group, and then relative expression values of tRNA of IRI group normalized to those of sham group were plotted. Note that benzonase was used to measure the abundance of indicated tRNAs which are mostly present inside EVs. EVs, extracellular vesicles. n.s. statistically non-significant; *p < 0.05.
