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Carraro, Andrea (2018) Individuazione di un potenziale profilo di miRNA, predittivi di rigetto subclinico in pazienti pediatrici trapiantati di rene. [Ph.D. thesis]

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Abstract (italian or english)

Kidney transplantation represents the optimal treatment for renal failure. The survival kidney graft rates about 83% (deceased donor) and 70% (living donor) in the pediatric transplanted patients at five years post transplantation.
The major cause of the delayed graft function and loss of the kidney is the rejection. Although the immunosuppressive therapy has reduced the onset of rejection, the endurance of the graft could further be improved, considering the life expectancy of a child. Currently, the standard procedure used in the diagnosis is the renal needle biopsy. Though the incidence of serious complications it has been reduced, needle biopsy remains an invasive practice for the patients and could lead to different risks such as infection or bleeding. Furthermore, kidney biopsy it is useful to improve kidney monitoring, however it does not avoid the histological damage.
The interest in new possible biomarkers useful in the prevention of kidney rejection is still notable. Among all the possible candidates, they have been identified the microRNAs (miRNAs), short noncoding RNA sequence of about 21 – 23 nucleotide long, involved in the mediation of several post transcriptional pathways. The miRNAs are involved in different processes such as cellular survival, development, differentiation, proliferation as well as to modulate the immunity. In the kidney, they seem to be involved in the regulation of renal development and in various physiological and pathological processes. Different studies have shown how the expression of miRNAs may change in biologic fluids and tissues in adult kidney transplanted patients. Therefore, miRNAs are currently being investigated as diagnostic/prognostic markers and possible therapeutic targets in kidney transplantation.
Another interesting source of biomarkers in the kidney rejection, it seems to be extracellular vesicles. Extracellular vesicles (EVs) are lipid bound nanoparticles release from all cell type. These particles act on target cells in different ways, including cell stimulation, transfer of proteins, lipids, genetic material, such as messenger RNA (mRNA) and miRNAs, to target cells, interacting even at a long distance.
Based on these evidences, we performed a retrospective miRNA screening on biopsies and serum extracellular vesicles samples of pediatric transplanted patients. Particularly, we selected a pool of twenty patients, 10 without histological diagnosis of rejection and 10 with histological diagnosis of subclinical rejection, at 1-year post transplantation. The miRNAs fraction showed a different range of concentration between tissue samples (0,7 e 7 ng/µl) and vesicles samples (0,06 e 0.52 ng/µl). The different levels of concentration are probably due to the isolation method of the vesicles, leading to a high dilution of the samples (resulting in the loss of a miRNA fraction). The miRNAs extracted were subsequently sequenced by NGS technology. The results of post-sequencing statistical analysis have highlighted five miRNAs (miR-142-3p, miR-142-5p, miR-101-3p, miR-106b-3p and miR-185-5p) significantly overexpressed on tissue’ samples of SCR patients (p-value <0.05%), whereas the same trend is not observed in serum EVs. Although the EVs samples did not show a different statistical expression of miRNAs, in the pool of vesicles, four out of the five miRNAs (miR-142-3p, miR-142-5p, miR-101-3p e miR-185-5p) expressed in the tissues of SCR patients, they were found.
It would be interesting to continue the research by checking the same miRNAs also in urinary EVs samples from pediatric transplanted kidney patients. Finally, this study could contribute to obtain a new laboratory test, useful to predict the onset of kidney rejection in pediatric patients and ameliorate the medical personalized therapy.

Abstract (a different language)

Il trapianto di rene rappresenta il trattamento ottimale per la cura dell’insufficienza renale terminale. La sopravvivenza dell’organo trapiantato nei pazienti pediatrici mostra un tasso percentuale a 5 anni del 83% nel caso di donatore vivente, e del 70%, nel caso di donatore cadavere.
La causa principale di disfunzione del graft con conseguente perdita è il rigetto renale. Sebbene la terapia immunosoppressiva abbia ridotto l’insorgenza di rigetto, la durata dell’organo trapiantato può ancora essere migliorata, considerando le aspettative di vita di un bambino. Attualmente la procedura standard usata per la diagnosi dello stato di salute del rene trapiantato è l’ago-biopsia renale, anche se rimane comunque una pratica invasiva per il paziente con possibile insorgenza, seppure in rari casi, di complicanze come infezioni o sanguinamento. Inoltre, la biopsia renale risulta utile nel monitoraggio del trapianto, ma non riesce a evitare l’insorgenza di danno istologico.
L’interesse verso nuovi possibili biomarcatori utili nella prevenzione del rigetto renale e tutt’ora rilevante. Tra i possibili candidati troviamo i microRNA (miRNA), corte sequenze di RNA non codificante lunghe 21 – 23 nt, coinvolti nella regolazione di diverse vie di segnalazione post-trascrizionali. I miRNA sono coinvolti in diversi processi come la sopravvivenza cellulare, lo sviluppo, la differenziazione, la proliferazione, e la modulazione della risposta immunitaria. A livello renale, sembrano avere un coinvolgimento nella regolazione dello sviluppo, in diversi processi fisiologici e patologici. Diversi studi hanno dimostrato come l’espressione dei miRNA possa variare nei fluidi biologici e in campioni tissutali di adulti trapiantati di rene. Per questo motivo, i miRNA sono attualmente studiati come possibili marker diagnostici/prognostici e potenziali targets terapeutici nel trapianto di rene.
Un'altra interessante risorsa di biomarker di rigetto renale, sembrano essere le vescicole extracellulari (EVs). Le EVs sono nanoparticelle sferiche costituite da strato fosfolipidico e rilasciate da tutte le tipologie cellulari. Queste particelle possono agire sulle cellule bersaglio in diversi modi, inclusa la stimolazione cellulare, il trasferimento di proteine, lipidi e materiale genetico, come RNA messaggero (mRNA) e miRNA, interagendo anche a lunga distanza.
Basandoci su questi dati si è deciso di effettuare uno studio retrospettivo dei miRNA espressi in campioni bioptici e vescicolari sierici di pazienti pediatrici trapiantati. In particolare, è stato selezionato un gruppo di 20 pazienti, 10 con diagnosi istologica normale e 10 con diagnosi di rigetto renale subclinico, ad 1 anno dal trapianto. La frazione di miRNA ottenuta aveva un range di concentrazione di 0,7 – 7 ng/µl nei campioni di tessuto renale e di 0,06 – 0,52 ng/µl nei campioni vescicolari. I miRNA estratti sono stati sequenziati con tecnologia NGS. I risultati dell’analisi statistica post sequenziamento hanno evidenziato 5 miRNA (miR-142-3p, miR-142-5p, miR-101-3p, miR-106b-3p and miR-185-5p) significativamente sovraespressi nei campioni tissutali di pazienti SCR (p-value <0,05), mentre lo stesso trend non è osservabile nelle EVs sieriche. Anche se i campioni vescicolari non hanno sottolineato differenze d’espressione dei miRNA statisticamente significative, nel pool identificato tramite sequenziamento sono stati rilevati 4 dei 5 miRNA sovraespressi nei tessuti dei CONT (miR-142-3p, miR-142-5p, miR-101-3p and miR-185-5p).
Sarebbe interessante proseguire la ricerca verificando gli stessi miRNA anche in campioni di EVs urinarie di pazienti pediatrici trapiantati di rene. Infine, questo studio può contribuire ad ottenere un test di laboratorio pratico e utile nella predizione d’insorgenza di rigetto renale in pazienti pediatrici e migliorare la terapia personalizzata.

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EPrint type:Ph.D. thesis
Tutor:Murer, Luisa
Supervisor:Negrisolo, Susanna
Ph.D. course:Ciclo 31 > Corsi 31 > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE SANITARIA
Data di deposito della tesi:28 November 2018
Anno di Pubblicazione:24 November 2018
Key Words:microRNA, vescicole extracellulari, pediatrico, trapianto di rene, rigetto subclinico
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/14 Nefrologia
Struttura di riferimento:Dipartimenti > Dipartimento di Salute della Donna e del Bambino
Codice ID:11413
Depositato il:07 Nov 2019 12:25
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