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Aims: Diabetic nephropathy is a major consequence of inflammation developing in type 1 diabetes, with interleukin-8 (IL-8)-CXCR1/2 axis playing a key role in kidney disease progression. In this study, we investigated the therapeutic potential of a CXCR1/2 non-competitive allosteric antagonist (Ladarixin) in preventing high glucose-mediated injury in human podocytes and epithelial cells differentiated from renal stem/progenitor cells (RSC) cultured as nephrospheres. Materials and Methods: We used human RSCs cultured as nephrospheres through a sphere-forming functional assay to investigate hyperglycemia-mediated effects on IL-8 signalling in human podocytes and tubular epithelial cells. Results: High glucose impairs RSC self-renewal, induces an increase in IL-8 transcript expression and protein secretion and induces DNA damage in RSC-differentiated podocytes, while exerting no effect on RSC-differentiated epithelial cells. Accordingly, the supernatant from epithelial cells or podocytes cultured in high glucose was able to differentially activate leucocyte-mediated secretion of pro-inflammatory cytokines, suggesting that the crosstalk between immune and non-immune cells may be involved in disease progression in vivo. Conclusions: Treatment with Ladarixin during RSC differentiation prevented high glucose-mediated effects on podocytes and modulated either podocyte or epithelial cell-dependent leucocyte secretion of pro-inflammatory cytokines, suggesting CXCR1/2 antagonists as possible pharmacological approaches for the treatment of diabetic nephropathy.

Tubulointerstitial fibrosis is observed in diabetic nephropathy. It is still debated whether tubular cells, undergoing epithelial-mesenchymal transition (EMT) in high glucose (HG) conditions, may contribute to interstitial fibrosis development. In this study, we investigated the phenotypic and molecular EMT-like changes and the alteration of inflammatory and fibrogenic secretome induced by HG in human primary tubular cell cultures. Taking advantage of this in vitro cell model composed of proximal and distal tubular cells, we showed that HG-treated tubular cells acquired a fibroblast-like morphology with increased cytoplasmic stress fibers, maintaining the expression of the epithelial markers specific of proximal and distal tubular cells. HG increased Snail1, miRNA210 and Vimentin mesenchymal markers, decreased N-cadherin expression and migration ability of primary tubular cells, while E-cadherin expression and focal adhesion distribution were not affected. Furthermore, HG treatment of tubular cells altered the inflammatory cytokine secretion creating a secretome able to enhance the proliferation and migration of fibroblasts. Our findings show that HG promotes an activated state of partial EMT in human tubular primary cells and induces a pro-inflammatory and pro-fibrogenic microenvironment, supporting the active role of tubular cells in diabetic nephropathy onset.

La fragilità è una sindrome geriatrica definita da un progressivo declino età-correlato di diverse facoltà fisiologiche, che si traduce in una ridotta funzionalità d’organo e in un’aumentata vulnerabilità in condizioni di stress. Fried e coll. propongono una definizione operativa delle fragilità. La letteratura riporta un’alta prevalenza di fragilità in individui affetti da insufficienza renale cronica. Tra questi soggetti, il rischio di sviluppare fragilità è aumentato di due/tre volte rispetto ai soggetti sani. Ad oggi, la definizione e la valutazione della fragilità in questi pazienti è ancora controversa. Tuttavia quello che si sa è che l’invecchiamento è un processo strettamente correlato alla ridotta capacità delle cellule staminali di auto-rinnovarsi e differenziarsi. Quest’alterazione delle funzioni delle cellule staminali può svolgere un ruolo chiave nella fisiopatologia delle malattie associate all’invecchiamento, inclusa la disfunzione renale. Il nostro gruppo ha identificato e isolato le cellule staminali renali adulte a partire da colture clonali di nefrosfere umane. All’interno di queste sono presenti cellule a diversa differenziazione e maturazione, tra queste anche le staminali identificate come PKHhigh/CD133+/CD24-, multipotenti e in grado di ripopolare scaffold renali decellularizzati. Date queste premesse l'obiettivo del progetto è valutare gli effetti di condizioni biologiche legate alla fragilità sul comportamento delle cellule staminali renali adulte umane e capire se tali condizioni sono in grado di esaurire il pool di cellule staminali e alterarne la funzione. In primo luogo abbiamo arruolato soggetti fragili, pre-fragili e non-fragili e giovani sani come controllo e raccolto i rispettivi plasmi e cellule mononucleate dal sangue periferico (PBMC). Sia nelle PBMC che nelle cellule staminali/progenitrici ematopoietiche circolanti abbiamo valutato il danno al DNA osservando una percentuale di cellule positive al danno statistic, Frailty is a geriatric syndrome that can be defined as an age-related progressive impairment of multiple physiological systems, resulting in a significantly reduced capacity to compensate for external stressors. Fried and colleagues proposed a phenotype characterization of frailty through five physical criteria, so this can be possible only after the onset of clinical manifestations without the possibility of a precocious diagnosis. Several studies report a high prevalence of frailty in both old and young individuals with kidney dysfunction, and this further increases with advancing age and progressive decline of renal function. Elderly individuals with chronic kidney disease (CKD) are two to three times more likely to be frail than those with normal renal function. However, the relationship between CKD and frailty is still unclear. The aging process can have adverse effects on stem cells; their self-renewal ability declines and their differentiation potential into the various cell types is altered. Aging-induced exhaustion and deterioration of stem cell pool and functions may play a key role in the pathophysiology of aging-associated diseases, including kidney dysfunction. Our group isolated a pure population of multipotent renal stem-like cells by a functional approach, taking advantage from the ability of renal stem cells (RSC) to grow as nephrospheres (NS). Investigating the expression of renal progenitor markers described in literature, our group identified in NS a homogeneous PKHhigh/CD133+/CD24- cell population displaying in vitro stem-cell properties, able to repopulate human decellularized renal scaffold and exhibiting multipotency. In this scenario, we tested whether in the organism of elderly and frail people there are biological conditions able to alter RSC behavior, justifying the high prevalence of chronic kidney dysfunction in the frail status and its severity. First, we recruited frail, pre-frail and non-frail subjects, and young subjects as controls

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.

The adipocyte-like morphology of clear cell renal cell carcinoma (ccRCC) cells results from a grade-dependent neutral lipid accumulation; however, the molecular mechanism and role in renal cancer progression have yet to be clarified. ccRCC shows a gene expression signature consistent with adipogenesis, and the phospholipid-binding protein annexin A3 (AnxA3), a negative regulator of adipocyte differentiation, is down-regulated in RCC and shows a differential expression pattern for two isoforms of 36 and 33 kDa. Using primary cell cultures and cell lines, we investigated the involvement of AnxA3 isoforms in lipid storage modulation of ccRCC cells. We found that the increased accumulation of lipids into ccRCC cells correlated with a decrease of the 36/33 isoform ratio. Treatment with adipogenic medium induced a significant increment of lipid storage in ccRCC cells that had a low 36-kDa AnxA3 expression and 36/33 ratio. The 36-kDa AnxA3 silencing in ccRCC cells increased lipid storage induced by adipogenic medium. These data suggest that 36-kDa AnxA3 negatively modulates the response to adipogenic treatment and may act as negative regulator of lipid storage in ccRCC cells. The subcellular distribution of AnxA3 in the cellular endocytic compartment suggests its involvement in modulation of vesicular trafficking, and it might serve as a putative mechanism of lipid storage regulation in ccRCC cells, opening novel translational outcomes.

Il meccanismo alla base della riparazione di un danno a cellule renali è ancora oggi oggetto di dibattito e potrebbe coinvolgere sia cellule terminalmente differenziate che l'esistenza di cellule staminali multipotenti quiescenti. Sfruttando il saggio di formazione di sfere e la tecnica del FACS sorting, il nostro gruppo ha isolato una popolazione di cellule marcate col clorante PKH26 che avessero caratteristiche di cellule staminali renali adulte (PKHhigh). In precedenza abbiamo valutato la capacità di queste cellule di differenziare in vitro in lineage epiteliale, podocitico ed endoteliale. Abbiamo anche dimostrato che la popolazione PKHhigh è eterogenea nella sua composizione e all'interno delle nefrosfere le cellule con capacità simil-staminali sono PKHhigh/CD133+/CD24- (RSC). Abbiamo recentemente pubblicato che le nostre cellule delle nefrosfere, che comprendono cellule PKHhigh e la loro progenie PKHlow/neg, sono in grado di ripopolare scaffold renali umani decellularizzati. Con questa ricerca, ora abbiamo come scopo: i) di dimostrare le capacità rigenerative delle cellule staminali PKHhigh, anche in assenza della loro progenie PKHlow/neg. ii) di trovare la signature molecolare delle RSC che, tra PKHhigh, sono le cellule con capacità staminali più ampie. Per raggiungere questi obiettivi, abbiamo messo in coltura cellule PKHhigh su scaffold acellulari per 30 giorni e le cellule delle strutture ripopolate sono state caratterizzate mediante immunofluorescenza sequenziale, utilizzando specifici marcatori di differenziamento. Alcune strutture hanno indicato un differenziamento terminale in lineage epiteliale tubulare prossimale o distale o in quello endoteliale. Solo poche strutture mostravano invece la coespressione di alcuni o di tutti i marcatori testati, suggerendo un fenotipo ancora immaturo. Per la comprensione della signature molecolare delle RSC, è stata eseguita l'analisi trascrittomica delle RSC stesse, della loro progenie PKHlow/neg e di colture p, The mechanism underlying the recovery of renal cell injury is still a matter of debate that concerns the involvement of fully differentiated cells, or the existence of quiescent scattered multipotent stem cells. By sphere forming assay and sorting, our group isolated a population of PKH26 most fluorescent cells with characteristics of adult renal stem-like cells (PKHhigh cells). We previously assessed the ability of PKHhigh cells to differentiate in vitro along epithelial, podocytic and endothelial lineages. We also demonstrated that PKHhigh population is heterogeneous in composition and within nephrospheres the cells with stem capacities are PKHhigh/CD133+/CD24- (RSC). We recently published that our nephrosphere cells, comprising PKHhigh cells and their PKHlow/neg progeny, are able to repopulate human decellularized renal scaffolds. With this research, we now aim: I) to prove the regenerative capabilities of PKHhigh stem-like cells, even in absence of their PKHlow/neg progeny. II) to find the molecular signature of RSC that among PKHhigh cells are those with the wider stem capacities. To reach these aims, we cultured isolated PKHhigh cells on acellular scaffolds for 30 days and the cells of the repopulated structures were characterized by sequential immunofluorescence using specific markers of differentiation. Some structures indicated a specific lineage differentiation into proximal and distal tubular epithelium and endothelium. Only few structures coexpressed some or all markers tested, indicating still immature phenotypes. For the disclosure of RSC molecular signature, transcriptomic analysis of RSC, of their PKHlow/neg progeny and of terminally differentiated primary cell cultures (PCC) was performed and differentially expressed genes (DEG) were evidenced. Bioinformatic Gene Set Enrichment Analysis suggested a renal immature status of our RSC, but different from embryonic stem cells. Crossing DEG lists potential markers were obtained and some of them were selec

Background. The management of modifiable risk factors exposure, in particular dyslipidemia, is the first line of intervention in preventing cardiovascular events. HDLs have been demonstrated to have anti-inflammatory and atheroprotective properties and, among HDLs, Apolipoprotein A-I (APOA-1), which promotes reverse cholesterol efflux, has been deeply investigated for the great therapeutic potential, particularly emphasized for the naturally occurring mutation APOA-1Milano (APOA-1M). Despite the high therapeutic potential of these molecules, their purification and delivery into the disordered organism is still limited by a very low efficiency in these processes. Aim. To develop an efficient system of production and delivery of APOA-1 muteins without need of purification. Methods and Results. Rice plants were genetically modified to express APOA-1M protein in their seeds. Protein extract from transgenic rice seeds (the ‘APOA-1M rice milk’) was tested for functionality in vitro on THP-1 macrophages exposed to oxLDL. Protein extract from wild type rice seeds (the ‘WT rice milk’) was used as control. The APOA-1M rice milk, but not the WT rice milk, significantly reduced expression of MCP-1 in oxLDL-loaded THP-1 macrophages in a dose-dependent manner and it promoted reverse cholesterol efflux in THP-1 macrophages. The lack of toxicity and the tolerability of the orally administered APOA-1M rice milk was evaluated in healthy mice. In an early-intermediate atherosclerotic mouse model (Apoe-/-mice fed with Western Diet for 8 weeks), 3 weeks of APOA-1M, but not the WT, rice milk treatment (15d, 5d/week, by oral gavage) significantly reduced area of lipid deposition and lipids concentration at aortic arch (en face analysis). Moreover, the APOA-1M, but not the WT, rice milk treatment reduced the hepatic CD68-positive cells and ameliorated the lipid management gene expression profile in liver of WD-fed Apoe-/-mice. Interestingly, all these findings were observed in mice still e