부산대학교 도서관

Kidney transplantation (KT) is the best therapeutic approach for chronic kidney diseases (CKD) leading to an irreversible kidney failure. However, the follow-up of kidney function is still delicate. Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies, including graft dysfunctions after KT. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Conversely, urine is an easily accessible, minimally invasive fluid. Thus, the information contained in this fluid, and particularly in urinary extracellular vesicles (uEVs), may be a potential source to unravel new biomarkers associated with renal pathologies. Nowadays, several methods have been described to enrich uEVs, although most of them render a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Therefore, the first aim of the present thesis is to evaluate size-exclusion chromatography (SEC) as a suitable method to isolate uEVs. Our results demonstrated that urine ultrafiltration followed by SEC is a suitable option to isolate uEVs, considerably reducing the presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches. The second aim of the thesis is to investigate whether uEV profiling can differentiate the state of the kidney of living donors (LD) from deceased donors (DD) and compared to normo-functional patients after KT (NFP). This end would be of interest to elucidate if the drug therapy administrated affects the kidneys and, ultimately, their uEVs composition. Among other differences, next generation sequencing revealed that miR-326 was found significantly over-represented in LD, and miR-7706 was found only present in NFP, which are related with apoptosis and cell survival, respectively. Moreover, 70 proteins were differentially over-expressed in LD compared to NFP. Of note, the majority of these proteins were related with cell metabolism, which could be related to the immunosuppressive therapy of NFP. To summarize, in this pilot study we found that RNA and protein profile are substantially different between LD, DD and NFP, although these differences did not have a significant impact in short term renal function (one year). Overall, the results and differential profile of protein and miRNAs described in this thesis between kidney donors and NFPs suggest that in future comparative studies in post-KT patients uEVs from NFPs are the appropriate control rather than uEVs from LDs. Finally, we have analysed the glycosylation pattern of uVEs of donors (LD and DD) and post-KT patients showing graft dysfunction. For what we think is the first time, we have described the presence of fucoses in the uVEs. Moreover, despite the reduced cohort of this pilot study, we describe some differences in the lectin-binding assays between donors and patients. Yet, these findings must be confirmed in further studies.