Urine-derived Renal Epithelial Cells from kidney transplanted patients: phenotype, immunomodulatory properties, and effect of the exposition to NGAL

During kidney transplant procedure transplanted organs can undergo ischaemia reperfusion phenomena, often associated with the onset of acute kidney damage, loss of kidney function and rejection. These events promote cell turnover to replace damaged cells and preserve kidney function, thus cells deriving from nephrons structures are highly voided in urine. Urine derived cells represents a promising cell source since they can be easily isolated and cultured. The aim of this project was to characterise Urine-derived Renal Epithelial Cells (URECs) from transplanted kidney and to evaluate how these cells react to the co-culture with immune cells. URECs expressed typical markers of kidney tubule epithelial cells (Cytokeratin and CD13), and a subpopulation of these cells expressed CD24 and CD133, which are markers of kidney epithelial progenitor cells. The expression of immunosuppressive molecules as HLA-G and CD73 was also observed. As matter of fact, during the co-culture with PBMCs, UREC suppressed the proliferation of CD4 and CD8 Lymphocytes and reduce the T helper 1 subset, while increasing the T regulatory counterpart. Also, preliminary data observed in this study indicated that the exposition to kidney damage associated molecule, such as NGAL, could significantly affect UREC viability and immunomodulatory capacity. These results add new information about the phenotype of urine cells obtained after kidney transplant and reveal that these cells show promising immunomodulatory properties, suggesting their potential application in personalized cell therapy approaches.

Progenitori endoteliali nei pazienti con Chronic Kidney Disease - Mineral and Bone Disorder (CKD-MBD) in fase uremica: effetti del trattamento con vitamina D.

L'insufficienza renale cronica (CKD) è associata ad un rischio cardiovascolare più elevato rispetto alla popolazione generale: fattori come uremia, stress ossidativo, età dialitica, infiammazione, alterazioni del metabolismo minerale e presenza di calcificazioni vascolari incidono fortemente sulla morbosità e mortalità per cause cardiovascolari nel paziente uremico. Diversi studi hanno verificato il coinvolgimento dei progenitori endoteliali (EPC) nella malattia aterosclerotica ed è stato dimostrato che esprimono osteocalcina, marcatore di calcificazione. Inoltre, nella CKD è presente una disfunzione in numero e funzionalità delle EPC. Attualmente, il ruolo delle EPC nella formazione delle calcificazioni vascolari nei pazienti in dialisi non è stato ancora chiarito. Lo scopo della tesi è quello di studiare le EPC prelevate da pazienti con CKD, al fine di determinarne numero e fenotipo. È stato anche valutato l'effetto del trattamento in vitro e in vivo con calcitriolo e paracalcitolo sulle EPC, dato il deficit di vitamina D dei pazienti con CKD: il trattamento con vitamina D sembra avere effetti positivi sul sistema cardiovascolare. Sono stati valutati: numero di EPC circolanti e la relativa espressione di osteocalcina e del recettore della vitamina D; morfologia e fenotipo EPC in vitro; effetti di calcitriolo e paracalcitolo sull’espressione di osteocalcina e sui depositi di calcio. I risultati dello studio suggeriscono che il trattamento con vitamina D abbia un effetto positivo sulle EPC, aumentando il numero di EPC circolanti e normalizzandone la morfologia. Sia calcitriolo che paracalcitolo sono in grado di ridurre notevolmente l’espressione di OC, mentre solo il paracalcitolo ha un effetto significativo sulla riduzione dei depositi di calcio in coltura. In conclusione, il trattamento con vitamina D sembra ridurre il potenziale calcifico delle EPC nell’uremia, aprendo nuove strade per la gestione del rischio cardiovascolare nei pazienti affetti da CKD.

A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Circulating Fibrocytes, their role in renal fibrosis and molecular pathways involved. Possible biomarkers of fibrogenesis in chronic kidney disease

Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

Modeling of clear cell sarcoma of the kidney of the pediatric age

Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor, characterized in 90% of cases by the presence of internal tandem duplications (ITDs) localized at the last exon of BCOR gene. BCOR protein constitute a core component of the non-canonical Polycomb Repressive Complex1 (PRC1.1), which performs a fundamental silencing activity. ITDs in the last BCOR exon at the level of PUFD domain have been identified in many tumor subtypes and could affect PCGF1 binding and the subsequent PRC1.1 activity, although the exact oncogenic mechanism of ITD remains poorly understood. This project has the objective of investigating the molecular mechanisms underlying the oncogenesis of CCSK, approaching the study with different methodologies. A first model in HEK-293 allowed to obtain important informations about BCOR functionality, suggesting that the presence of ITD generates an altered activity which is very different from a loss-of-function. It has also been observed that BCOR function within the PRC1.1 complex varies with different ITDs. Moreover, it allowed the identification of molecular signatures evoked by the presence of BCOR-ITD, including its role in extracellular matrix interactions and invasiveness promotion. The parallel analysis of WTS data from 8 CCSK cases permitted the identification of a peculiar signature for metastatic CCSKs, highlighting a 20-fold overexpression of FGF3. This factor promoted a significant increase in invasive ability in the cellular model. In order to study BCOR-ITD effects over cell stemness and differentiation, an inducible model is being obtained in H1 cells. This way, it will be possible to study the functionality of BCOR-ITD in a context more similar to the origin of CCSKs, evaluating both the specific interactome and phenotypic consequences caused by the mutation.