ESPN 50th Annual Meeting

ESPN 2017


 
URINARY EXOSOMES ISOLATION: DIFFERENT METHODS TO DISCOVER NOVEL BIOMARKERS OF KIDNEY REJECTION
ANDREA CARRARO 1 SUSANNA NEGRISOLO 1 GIULIA FREGONESE 1 ELISA BENETTI 1 ANNA MARIA TOLOMEO 2 LUISA MURER 1

1- UNIVERSITY HOSPITAL OF PADOVA, PEDIATRIC NEPHROLOGY, DIALYSIS AND TRANSPLANT UNIT, DEPARTMENT OF WOMENS AND CHILDRENS HEALTH, ITALY
2- UNIVERSITY HOSPITAL OF PADOVA, DEPARTMENT OF WOMENS AND CHILDRENS HEALTH, ITALY
 
Introduction:

Exosomes are lipid membrane-bound nanoparticles (40–150 nm of size) released from different cells type. They could carry different types of cargo (e.g. miRNA, proteins) that reflect the physiopathological status of the cells and/or organ they originated from. For instance, the exosomes present in the urine called Urinary Exosomes (UExs) arise from all the different nephron cells. Thus, an accurate characterization of their content could be helpful to identify novel reliable non-invasive biomarkers for kidney allograft injury. However, due to their low amount, UExs concentration and characterization remain a challenge. This study aims to identify the most efficient UExs isolation method both for RNA profiling and proteomic analysis to discover novel renal transplant rejection biomarkers.

Material and methods:

UExs were isolated from urine samples using four different methods: three commercial kits (Norgen, ExoQuick and Qiagen) and the ultrafiltration. UExs were quantified by qNano. Total RNA, included small RNA species, was extracted using column kit. Biochemical assay was applied to extract proteins. The miRNA and proteins integrity and concentration were evaluated by Agilent Bioanalyzer 2100.

Results:

The UExs isolated with the four methods differ in raw concentration (3.5x108–3.5x1012) and size (90–130 nm). qNano analysis showed that Qiagen kit was the most efficient isolation method for UExs. However, Agilent Bioanalyzer RNA analysis emphasized that the highest amount of miRNAs was obtained using Norgen Kit. Same kind of experiments are currently ongoing for the analysis of the UExs proteins content.

Conclusions:

Based on our results, all four methods yielded UExs with a high variability in number and diameter. Furthermore, Norgen kit is the most productive method for miRNA UEXs isolation. The next step of this study will be the evaluation of UExs characterization in correlation with the different outcome observed in the pediatric population recruited in our renal transplanted center.