ABSTRACT
Objective:To compare the expression level of exosomal miR-503 in peritoneal dialysis effluent (PDE) from patients of different peritoneal transport characteristics, predict the target genes of miR-503 and provide bioinformatic data for researches of peritoneal transport characteristics.Methods:Twenty-four stable peritoneal dialysis (PD) patients were selected and divided into high transport group (H group, n=12) and low transport group (L group, n=12) according to the results of peritoneal equilibration tests (PET). The 500 ml PDE that was left on the patient's abdomen overnight was collected and concentrated using ultrafiltration cell. Exosomes in PDE were resuspended in phosphate buffered saline (PBS) after ultracentrifugation and the characteristics of PDE exosomes were identified by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), Western blotting and fluorescent staining. MicroRNAs were extracted from PDE exosomes. The expression levels of PDE exosomal miR-503 in the two groups were detected by quantitative real-time PCR. Then the relations between the relative quantity of PDE exosomal miR-503 and PET values or 24 h ultrafiltration volume (UF) were analyzed. Targetscan and miRDB databases were used to predict the target genes of miR-503. Gene ontology (GO) functional enrichment and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway analysis were relied on DAVID (https://david.ncifcrf.gov/). Results:The exosomes in PDE showed a round and cup-shaped morphology under TEM, and the diameters were approximately 100 nm measured by NTA. The specific biomarkers of exosomes, CD63, CD81 and heat shock protein -70 (HSP-70) were all detected by Western blotting. The internalization and uptake of the exosomes was observed after fluorescent staining. The relative expression level of PDE exosomal miR-503 in H group was found to be significantly higher than that in L group ( P=0.002), and the relative quantity of PDE exosomal miR-503 was significantly positively correlated with PET values ( r=0.547, P=0.006), but not 24 h UF ( r=-0.297, P=0.159). There were 156 target genes of miR-503 in total that could be predicted by two different databases at the same time. GO analysis of these 156 target genes was mainly focused on kinase binding, regulation of protein modification and catabolic process as well as regulation of epithelial cell proliferation. KEGG enriched many tumor associated or classical signaling pathways, including transforming growth factor-β (TGF-β) signaling pathway and vascular endothelial growth factor (VEGF) signaling pathway. The prediction showed that vascular endothelial growth factor A (VEGFA) was a direct target gene of miR-503 and it was also related to many proteins involved in fibrosis mechanism. Conclusions:The expression level of PDE exosomal miR-503 is significantly higher in H group, and positively correlates with PET values, which may regulate the angiogenesis of peritoneal vessels by targeting VEGFA.
ABSTRACT
Objective To establish blood purification system for rats and provide a safe and reliable experimental platform for further research of blood purification. Methods The right carotid artery and the contralateral jugular vein of adult male Sprague?Dawley rats were cannulated to creat vascular access for blood purification, by which continuous arteriovenous hemofiltration blood purification system was established. Blood flow, substitution fluid flow and ultrafiltration rate were regulated by rotary mini?pumps. Blood purification therapy continued for 4 hours on the basis of maintained anesthesia and effective anticoagulation. The safety of continuous arteriovenous hemofiltration blood purification systems was evaluated by comparing the arterial blood gas, electrolyte indexes and blood glucose during the blood purification therapy. Closely monitoring the vital signs of rats, such as blood pressure and heart rate, and observing whether there were any side effects, such as massive haemorrhage, thrombogenesis and gas embolism in the therapeutic process. Results There were no obvious changes of arterial blood gas, electrolyte indexes and blood glucose during the blood purification therapy (P>0.05). The vital signs did not fluctuate acutely before and after the blood purification therapy (P>0.05). The incidence rate of side effects was very low. Conclusions Continuous arteriovenous hemofiltration blood purification system had no obvious adverse effects on healthy rats. Our blood purification system for rats appears to be safe and reliable.