Introduction: Delayed graft function (DGF) is a common complication after deceased donor kidney transplantation (KT) which affects short and long-term outcome. Currently available biomarkers in perfusate lack sensitivity in predicting graft outcome.
Our group has already set up a protocol to determine miRNAs in preservation solution during graft perfusion and a panel was selected based on modulation of expression and functional relevance. New normalization guidelines in miRNAs analysis have been recently established.
The aim of the study was to validate the selected miRNAs panel according to those renewed normalization guidelines as a way of results support.
Material and Methods: We conducted a prospective cohort study of graft dysfunction in KT from ECD. Ethical approval was obtained from Ethics Review Board. As a discovery cohort, a total of 8 samples selected according to immediate and graft function at 1 year were analyzed. miRNAs were previously detected and quantified by Real-Time PCR performed using SYBR Green and specific LNA probes for each miRNA of interest (Exiqon) as well as exogenous Spike- in as technical control. Renewed normalization guidelines involved the use of the average of all miRNAs expression. The statistical significant differences were observed by means of GenEX v6 Enterprise software from Exiqon.
Results: miRNAs screening experiment was performed in order to determine the expression of 762 different miRNAs in the preservation solutions from human allografts, exhibiting DGF and/or altered renal function after one year of transplantation. After discarding miRNAs CT differences due to technical variation, initial normalization was performed using Spike-in (UniSP2) levels. Data analysis by SPSS indicate that miR-486, miR-18a, miR20a, miR363-3p, miR144-3p, miR454-3p, mir223 3p, miR142-5p, miR502-3p, miR144-3p and miR144-5p discriminate between the DGF appearance after transplantation. Moreover, miR130a-3p, miR-30e-5p, miR-324-3p were associated to the diminished renal function after one year (GRF < 30).
A second normalization analysis using GenEX v6 Enterprise software from Exiqon, using the average of all miRNAs expression as normalization parameter was performed. This second analysis confirm that 486-5p, 144-3p, 142-5p, 144-5p, are again associated to DGF appearance. Two new miRNAs, 199a-5p and 497-5p are now also associated to DGF appearance. Regarding diminished renal function after a year, 148b-3p and 20a-5p are significant.
Conclusions: We have identified miRNAs detected in renal preservation solution that can be associated to DGF appearance after transplantation. Interestingly, some miRNAs detected also in preservation solution could indicate allograft long term outcome, since they associate to diminished renal function after one year. Analysis compliance with new normalization international guidelines confirm the significance of most of the miRNAs previously identified.