Refractory IgA Nephropathy: A Challenge for Future Nephrologists.

IgA nephropathy (IgAN) represents the most prevalent form of primary glomerulonephritis, and, on a global scale, it ranks among the leading culprits behind end-stage kidney disease (ESKD). Presently, the primary strategy for managing IgAN revolves around optimizing blood pressure and mitigating proteinuria. This is achieved through the utilization of renin-angiotensin system (RAS) inhibitors, namely, angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARBs). As outlined by the KDIGO guidelines, individuals who continue to show a persistent high risk of progressive ESKD, even with comprehensive supportive care, are candidates for glucocorticoid therapy. Despite these therapies, some patients have a disease refractory to treatment, defined as individuals that present a 24 h urinary protein persistently >1 g after at least two rounds of regular steroids (methylprednisolone or prednisone) and/or immunosuppressant therapy (e.g., mycophenolate mofetil), or who do not tolerate regular steroids and/or immunosuppressant therapy. The aim of this Systematic Review is to revise the current literature, using the biomedical database PubMed, to investigate possible therapeutic strategies, including SGLT2 inhibitors, endothelin receptor blockers, targeted-release budesonide, B cell proliferation and differentiation inhibitors, fecal microbiota transplantation, as well as blockade of complement components.

Beneficial effects of recombinant CER-001 high-density lipoprotein infusion in sepsis: results from a bench to bedside translational research project.

BACKGROUND: Sepsis is characterized by a dysregulated immune response and metabolic alterations, including decreased high-density lipoprotein cholesterol (HDL-C) levels. HDL exhibits beneficial properties, such as lipopolysaccharides (LPS) scavenging, exerting anti-inflammatory effects and providing endothelial protection. We investigated the effects of CER-001, an engineered HDL-mimetic, in a swine model of LPS-induced acute kidney injury (AKI) and a Phase 2a clinical trial, aiming to better understand its molecular basis in systemic inflammation and renal function. METHODS: We carried out a translational approach to study the effects of HDL administration on sepsis. Sterile systemic inflammation was induced in pigs by LPS infusion. Animals were randomized into LPS (n = 6), CER20 (single dose of CER-001 20 mg/kg; n = 6), and CER20 × 2 (two doses of CER-001 20 mg/kg; n = 6) groups. Survival rate, endothelial dysfunction biomarkers, pro-inflammatory mediators, LPS, and apolipoprotein A-I (ApoA-I) levels were assessed. Renal and liver histology and biochemistry were analyzed. Subsequently, we performed an open-label, randomized, dose-ranging (Phase 2a) study included 20 patients with sepsis due to intra-abdominal infection or urosepsis, randomized into Group A (conventional treatment, n = 5), Group B (CER-001 5 mg/kg BID, n = 5), Group C (CER-001 10 mg/kg BID, n = 5), and Group D (CER-001 20 mg/kg BID, n = 5). Primary outcomes were safety and efficacy in preventing AKI onset and severity; secondary outcomes include changes in inflammatory and endothelial dysfunction markers. RESULTS: CER-001 increased median survival, reduced inflammatory mediators, complement activation, and endothelial dysfunction in endotoxemic pigs. It enhanced LPS elimination through the bile and preserved liver and renal parenchyma. In the clinical study, CER-001 was well-tolerated with no serious adverse events related to study treatment. Rapid ApoA-I normalization was associated with enhanced LPS removal and immunomodulation with improvement of clinical outcomes, independently of the type and gravity of the sepsis. CER-001-treated patients had reduced risk for the onset and progression to severe AKI (stage 2 or 3) and, in a subset of critically ill patients, a reduced need for organ support and shorter ICU length of stay. CONCLUSIONS: CER-001 shows promise as a therapeutic strategy for sepsis management, improving outcomes and mitigating inflammation and organ damage. TRIAL REGISTRATION: The study was approved by the Agenzia Italiana del Farmaco (AIFA) and by the Local Ethic Committee (N° EUDRACT 2020-004202-60, Protocol CER-001- SEP_AKI_01) and was added to the EU Clinical Trials Register on January 13, 2021.

The Long Non-coding RNA HOTAIR Controls the Self-renewal, Cell Senescence, and Secretion of Anti-aging Protein α-Klotho in Human Adult Renal Progenitor Cells.

The long non-coding RNAs (lncRNA) play an important role in several biological processes, including some renal diseases. Nevertheless, little is known about lncRNA that are expressed in the healthy kidneys and involved in renal cell homeostasis and development, and even less is known about lncRNA involved in the maintenance of human adult renal stem/progenitor cells (ARPCs) that have been shown to be very important for renal homeostasis and repair processes. Through a whole-genome transcriptome screening, we found that the HOTAIR lncRNA is highly expressed in renal progenitors and potentially involved in cell cycle and senescence biological processes. By CRISPR/Cas9 genome editing, we generated HOTAIR knockout ARPC lines and established a key role of this lncRNA in ARPC self-renewal properties by sustaining their proliferative capacity and limiting the apoptotic process. Intriguingly, the HOTAIR knockout led to the ARPC senescence and to a significant decrease in the CD133 stem cell marker expression which is an inverse marker of ARPC senescence and can regulate renal tubular repair after the damage. Furthermore, we found that ARPCs expressed high levels of the α-Klotho anti-aging protein and especially 2.6-fold higher levels compared to that secreted by renal proximal tubular cells (RPTECs). Finally, we showed that HOTAIR exerts its function through the epigenetic silencing of the cell cycle inhibitor p15 inducing the trimethylation of the histone H3K27. Altogether, these results shed new light on the mechanisms of regulation of these important renal cells and may support the future development of precision therapies for kidney diseases.

The Human Virome and Its Crosslink with Glomerulonephritis and IgA Nephropathy.

The prokaryotic, viral, fungal, and parasitic microbiome exists in a highly intricate connection with the human host. In addition to eukaryotic viruses, due to the existence of various host bacteria, phages are widely spread throughout the human body. However, it is now evident that some viral community states, as opposed to others, are indicative of health and might be linked to undesirable outcomes for the human host. Members of the virome may collaborate with the human host to retain mutualistic functions in preserving human health. Evolutionary theories contend that a particular microbe's ubiquitous existence may signify a successful partnership with the host. In this Review, we present a survey of the field's work on the human virome and highlight the role of viruses in health and disease and the relationship of the virobiota with immune system control. Moreover, we will analyze virus involvement in glomerulonephritis and in IgA nephropathy, theorizing the molecular mechanisms that may be responsible for the crosslink with these renal diseases.

Pulsed Electromagnetic Fields Induce Skeletal Muscle Cell Repair by Sustaining the Expression of Proteins Involved in the Response to Cellular Damage and Oxidative Stress.

Pulsed electromagnetic fields (PEMF) are employed as a non-invasive medicinal therapy, especially in the orthopedic field to stimulate bone regeneration. However, the effect of PEMF on skeletal muscle cells (SkMC) has been understudied. Here, we studied the potentiality of 1.5 mT PEMF to stimulate early regeneration of human SkMC. We showed that human SkMC stimulated with 1.5 mT PEMF for four hours repeated for two days can stimulate cell proliferation without inducing cell apoptosis or significant impairment of the metabolic activity. Interestingly, when we simulated physical damage of the muscle tissue by a scratch, we found that the same PEMF treatment can speed up the regenerative process, inducing a more complete cell migration to close the scratch and wound healing. Moreover, we investigated the molecular pattern induced by PEMF among 26 stress-related cell proteins. We found that the expression of 10 proteins increased after two consecutive days of PEMF stimulation for 4 h, and most of them were involved in response processes to oxidative stress. Among these proteins, we found that heat shock protein 70 (HSP70), which can promote muscle recovery, inhibits apoptosis and decreases inflammation in skeletal muscle, together with thioredoxin, paraoxonase, and superoxide dismutase (SOD2), which can also promote skeletal muscle regeneration following injury. Altogether, these data support the possibility of using PEMF to increase SkMC regeneration and, for the first time, suggest a possible molecular mechanism, which consists of sustaining the expression of antioxidant enzymes to control the important inflammatory and oxidative process occurring following muscle damage.

Extracellular vesicles derived from patients with antibody-mediated rejection induce tubular senescence and endothelial to mesenchymal transition in renal cells.

Extracellular vesicles (EV) are emerging mediators in several diseases. However, their role in the pathophysiology of antibody-mediated allograft rejection (AMR) has been poorly investigated. Here, we investigated the role of EV isolated from AMR patients in inducing tubular senescence and endothelial to mesenchymal transition (EndMT) and analyzed their miRNA expression profile. By multiplex bead flow cytometry, we characterized the immunophenotype of plasma AMR-derived EV and found a prevalent platelet and endothelial cell origin. In vitro, AMR-derived EV induced tubular senescence by upregulating SA-ß Gal and CDKN1A mRNA. Furthermore, AMR-derived EV induced EndMT. The occurrence of tubular senescence and EndMT was confirmed by analysis of renal biopsies from the same AMR patients. Moreover, AMR-derived EV induced C3 gene upregulation and CFH downregulation in tubular epithelial cells, with C4d deposition on endothelial cells. Interestingly, RNase-mediated digestion of EV cargo completely abrogated tubular senescence and EndMT. By microarray analysis, miR-604, miR-515-3p, miR-let-7d-5p, and miR-590-3p were significantly upregulated in EV from AMR group compared with transplant controls, whereas miR-24-3p and miR-29a-3p were downregulated. Therefore, EV-associated miRNA could act as active player in AMR pathogenesis, unraveling potential mechanisms of accelerated graft senescence, complement activation and early fibrosis that might lead to new therapeutic intervention.

Toll-Like Receptors in Stem/Progenitor Cells.

One of the bridges that control the cross-talk between the innate and adaptive immune systems is toll-like receptors (TLRs). TLRs interact with molecules shared and maintained by the source pathogens, but also with endogenous molecules derived from injured tissues (damage/danger-associated molecular patterns - DAMPs). This is likely why some kinds of stem/progenitor cells (SCs) have been found to express TLRs. The role of TLRs in regulating basal motility, proliferation, processes of differentiation, self-renewal, and immunomodulation has been demonstrated in these cells. In this book chapter, we will discuss the many different functions assumed by the TLRs in SCs, pointing out that, depending on the context and the type of ligands they perceive, they may have different effects. In addition, the role of TLR in SC's response to specific tissue damage and in reparative processes will be addressed, as well as how the discovery of molecules mediating TLR signaling's differential function may be decisive for the development of new therapeutic strategies. Given the available studies on TLRs in SCs, the significance of TLRs in sensing an injury to stem/progenitor cells and evaluating their action and reparative activity, which depends on the circumstances, will be discussed here. It could also be possible that SCs used in therapy could theoretically be exposed to TLR ligands, which could modulate their in vivo therapeutic potential. In this context, we need to better understand the mechanisms of action of TLRs on SCs and learn how to regulate these receptors and their downstream pathways in a precise way in order to modulate SC proliferation, survival, migration, and differentiation in the pathological environment. In this way, cell therapy may be strengthened and made safer in the future.

High levels of gut-homing immunoglobulin A+ B lymphocytes support the pathogenic role of intestinal mucosal hyperresponsiveness in immunoglobulin A nephropathy patients.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis. The role of the microbiota and mucosal immunity in the pathogenesis of IgAN remains a key element. To date, the hypothetical relationship between commensal bacteria, elevated tumour necrosis factor (TNF) superfamily member 13 [also known as B-cell activating factor (BAFF)] levels, perturbed homoeostasis of intestinal-activated B cells and intestinal IgA class switch has not been clearly shown in IgAN patients. METHODS: We studied the intestinal-renal axis connections, analysing levels of BAFF, TNF ligand superfamily member 13 (APRIL) and intestinal-activated B cells in IgAN patients, healthy subjects (HSs) and patients with non-IgA glomerulonephritides. RESULTS: IgAN patients had increased serum levels of BAFF cytokine, correlating with higher amounts of five specific microbiota metabolites, and high APRIL cytokine serum levels. We also found that subjects with IgAN have a higher level of circulating gut-homing (CCR9+ ß7 integrin+) regultory B cells, memory B cells and IgA+ memory B cells compared with HSs. Finally, we found that IgAN patients had high levels of both total plasmablasts (PBs) and intestinal-homing PBs. Interestingly, PBs significantly increased in IgAN but not in patients with other glomerulonephritides. CONCLUSIONS: Our results demonstrate a significant difference in the amount of intestinal-activated B lymphocytes between IgAN patients and HSs, confirming the hypothesis of the pathogenic role of intestinal mucosal hyperresponsiveness in IgAN. The intestinal-renal axis plays a crucial role in IgAN and several factors may contribute to its complex pathogenesis and provide an important area of research for novel targeted therapies to modulate progression of the disease.

Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.

Endothelial dysfunction is a hallmark of LPS-induced acute kidney injury (AKI). Endothelial cells (ECs) acquired a fibroblast-like phenotype and contributed to myofibroblast generation through the endothelial-to-mesenchymal transition (EndMT) process. Of note, human adult renal stem/progenitor cells (ARPCs) enhance the tubular regenerative mechanism during AKI but little is known about their effects on ECs. Following LPS exposure, ECs proliferated, decreased EC markers CD31 and vascular endothelial cadherin, and up-regulated myofibroblast markers, collagen I, and vimentin. The coculture with ARPCs normalized the EC proliferation rate and abrogated the LPS-induced EndMT. The gene expression analysis showed that most of the genes modulated in LPS-stimulated ARPCs belong to cell activation and defense response pathways. We showed that the ARPC-specific antifibrotic effect is exerted by the secretion of CXCL6, SAA4, and BPIFA2 produced after the anaphylatoxin stimulation. Next, we investigated the molecular signaling that underlies the ARPC protective mechanism and found that renal progenitors diverge from differentiated tubular cells and ECs in myeloid differentiation primary response 88-independent pathway activation. Finally, in a swine model of LPS-induced AKI, we observed that activated ARPCs secreted CXCL6, SAA4, and BPIFA2 as a defense response. These data open new perspectives on the treatment of both sepsis- and endotoxemia-induced AKI, suggesting an underestimated role of ARPCs in preventing endothelial dysfunction and novel strategies to protect the endothelial compartment and promote kidney repair.-Sallustio, F., Stasi, A., Curci, C., Divella, C., Picerno, A., Franzin, R., De Palma, G., Rutigliano, M., Lucarelli, G., Battaglia, M., Staffieri, F., Crovace, A., Pertosa, G. B., Castellano, G., Gallone, A., Gesualdo, L. Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.

Adult Renal Stem/Progenitor Cells Can Modulate T Regulatory Cells and Double Negative T Cells.

Adult Renal Stem/Progenitor Cells (ARPCs) have been recently identified in the human kidney and several studies show their active role in kidney repair processes during acute or chronic injury. However, little is known about their immunomodulatory properties and their capacity to regulate specific T cell subpopulations. We co-cultured ARPCs activated by triggering Toll-Like Receptor 2 (TLR2) with human peripheral blood mononuclear cells for 5 days and 15 days and studied their immunomodulatory capacity on T cell subpopulations. We found that activated-ARPCs were able to decrease T cell proliferation but did not affect CD8+ and CD4+ T cells. Instead, Tregs and CD3+ CD4- CD8- double-negative (DN) T cells decreased after 5 days and increased after 15 days of co-culture. In addition, we found that PAI1, MCP1, GM-CSF, and CXCL1 were significantly expressed by TLR2-activated ARPCs alone and were up-regulated in T cells co-cultured with activated ARPCs. The exogenous cocktail of cytokines was able to reproduce the immunomodulatory effects of the co-culture with activated ARPCs. These data showed that ARPCs can regulate immune response by inducing Tregs and DN T cells cell modulation, which are involved in the balance between immune tolerance and autoimmunity.

Do thermal treatments influence the ultrafast opto-thermal processes of eumelanin?

After light absorption, melanin converts very rapidly the energy gained into heat. The time scale of this process ranges from tens of femtoseconds to a few nanoseconds. Femtosecond transient absorption allows for exploration of such photo-induced carrier dynamics to observe the de-excitation pathways of the biological complex. Here, we report on the ultrafast relaxation of suspensions of Sepia melanin in DMSO at room temperature using a femtosecond broadband pump and probe technique by photoexciting in the UV and probing in the entire visible range. In particular, we focus on the possible role that different heat treatments, performed in the temperature range 30-80 °C  might have on the relaxation of charge carriers photogenerated by UV radiation in such suspensions. Experimental data indicate that in all the investigated suspensions, photoexcited carriers always follow a tri-exponential route to relaxation. Moreover, we find that the relaxation time constants are essentially the same in all cases, within the experimental error. We take this as evidence that all the investigated suspensions essentially exhibit the same relaxation dynamics, regardless of the temperature at which the heat treatment has been performed and of the heat-induced denaturation of the proteinaceous compounds bound to the photoactive pigment. Our experiments represent a significant step towards the understanding of the stability of melanin with respect to temperature changes.

The Heterogeneity of Renal Stem Cells and Their Interaction with Bio- and Nano-materials.

For a long time, the kidney has been considered incapable of regeneration. Instead, in recent years, studies have supported the existence of heterogeneity of renal stem/progenitor cells with the ability to regenerate both glomerular and tubular epithelial cells. Indeed, several studies evidence that renal progenitor cells, releasing chemokines, growth factors, microvesicles, and transcription factors through paracrine mechanisms, can induce tissue regeneration and block pathological processes of the kidney. In this chapter the potentiality of the kidney regenerative processes is considered and reviewed, and the main classes of stem/progenitor cells that might contribute to the renal tissue renewal is analyzed. Moreover, we evaluate the role of biomaterials in the regulation of cellular functions, specifically addressing renal stem/progenitor cells. Materials can be synthesized and tailored in order to recreate a finely structured microenvironment (by nanostructures, nanofibers, bioactive compounds, etc.) with which the cells can interact actively. For instance, by patterning substrates in regions that alternately promote or prevent protein adsorption, cell adhesion and spreading processes can be controlled in space. We illustrate the potentiality of nanotechnologies and engineered biomaterials in affecting and enhancing the behavior of renal stem/progenitor cells. Although there are still many challenges for the translation of novel therapeutics, advances in biomaterials and nanomedicine have the potential to drastically change the clinical and therapeutic landscape, even in combination with stem cell biology.

A transcriptomics study of hereditary angioedema attacks.

BACKGROUND: Hereditary angioedema (HAE) caused by C1-inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. OBJECTIVE: We sought to explore the involvement of other putative genes in edema formation. METHODS: We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. RESULTS: Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real-time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. CONCLUSIONS: Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE.

A New Vision of IgA Nephropathy: The Missing Link.

IgA Nephropathy (IgAN) is a primary glomerulonephritis problem worldwide that develops mainly in the 2nd and 3rd decade of life and reaches end-stage kidney disease after 20 years from the biopsy-proven diagnosis, implying a great socio-economic burden. IgAN may occur in a sporadic or familial form. Studies on familial IgAN have shown that 66% of asymptomatic relatives carry immunological defects such as high IgA serum levels, abnormal spontaneous in vitro production of IgA from peripheral blood mononuclear cells (PBMCs), high serum levels of aberrantly glycosylated IgA1, and an altered PBMC cytokine production profile. Recent findings led us to focus our attention on a new perspective to study the pathogenesis of this disease, and new studies showed the involvement of factors driven by environment, lifestyle or diet that could affect the disease. In this review, we describe the results of studies carried out in IgAN patients derived from genomic and epigenomic studies. Moreover, we discuss the role of the microbiome in the disease. Finally, we suggest a new vision to consider IgA Nephropathy as a disease that is not disconnected from the environment in which we live but influenced, in addition to the genetic background, also by other environmental and behavioral factors that could be useful for developing precision nephrology and personalized therapy.

LPS-Binding Protein Modulates Acute Renal Fibrosis by Inducing Pericyte-to-Myofibroblast Trans-Differentiation through TLR-4 Signaling.

During sepsis, the increased synthesis of circulating lipopolysaccharide (LPS)-binding protein (LBP) activates LPS/TLR4 signaling in renal resident cells, leading to acute kidney injury (AKI). Pericytes are the major source of myofibroblasts during chronic kidney disease (CKD), but their involvement in AKI is poorly understood. Here, we investigate the occurrence of pericyte-to-myofibroblast trans-differentiation (PMT) in sepsis-induced AKI. In a swine model of sepsis-induced AKI, PMT was detected within 9 h from LPS injection, as evaluated by the reduction of physiologic PDGFRß expression and the dysfunctional α-SMA increase in peritubular pericytes. The therapeutic intervention by citrate-based coupled plasma filtration adsorption (CPFA) significantly reduced LBP, TGF-ß, and endothelin-1 (ET-1) serum levels, and furthermore preserved PDGFRß and decreased α-SMA expression in renal biopsies. In vitro, both LPS and septic sera led to PMT with a significant increase in Collagen I synthesis and α-SMA reorganization in contractile fibers by both SMAD2/3-dependent and -independent TGF-ß signaling. Interestingly, the removal of LBP from septic plasma inhibited PMT. Finally, LPS-stimulated pericytes secreted LBP and TGF-ß and underwent PMT also upon TGF-ß receptor-blocking, indicating the crucial pro-fibrotic role of TLR4 signaling. Our data demonstrate that the selective removal of LBP may represent a therapeutic option to prevent PMT and the development of acute renal fibrosis in sepsis-induced AKI.

Omics studies for comprehensive understanding of immunoglobulin A nephropathy: state-of-the-art and future directions.

Immunoglobulin A nephropathy (IgAN) is the most common worldwide primary glomerulonephritis with a strong autoimmune component. The disease shows variability in both clinical phenotypes and endpoints and can be potentially subdivided into more homogeneous subtypes through the identification of specific molecular biomarkers. This review focuses on the role of omics in driving the identification of potential molecular subtypes of the disease through the integration of multilevel data from genomics, transcriptomics, epigenomics, proteomics and metabolomics. First, the identification of molecular biomarkers, including mapping of the full spectrum of common and rare IgAN risk alleles, could permit a more precise stratification of IgAN patients. Second, the analysis of transcriptomic patterns and their modulation by epigenetic factors like microRNAs has the potential to increase our understanding in the pathogenic mechanisms of the disease. Third, the specificity of urinary proteomic and metabolomic signatures and the understanding of their functional relevance may contribute to the development of new non-invasive biomarkers for a better molecular characterization of the renal damage and its follow-up. All these approaches can give information for targeted therapeutic decisions and will support novel clinical decision making. In conclusion, we offer a framework of omic studies and outline barriers and potential solutions that should be used for improving the diagnosis and treatment of the disease. The ongoing decade is exploiting novel high-throughput molecular technologies and computational analyses for improving the diagnosis (precision nephrology) and treatment (personalized therapy) of the IgAN subtypes.

In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets.

The administration of Everolimus (EVE), a mTOR inhibitor used in transplantation and cancer, is often associated with adverse effects including pulmonary fibrosis. Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. To improve our knowledge, primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 h. EMT markers (α-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. Our results revealed a complex biological network implicated in EVE-related pulmonary fibrosis and underlined new potential disease biomarkers and therapeutic targets.

Epigenetic dysregulation in neuroblastoma: A tale of miRNAs and DNA methylation.

In neuroblastoma, the epigenetic landscape is more profoundly altered in aggressive compared to lower grade tumors and the concomitant hypermethylation of many genes, defined as "methylator phenotype", has been associated with poor outcome. DNA methylation can interfere with gene expression acting at distance through the methylation or demethylation of the regulatory regions of miRNAs. The multiplicity of miRNA targets may result in the simultaneous alteration of many biological pathways like cell proliferation, apoptosis, migration and differentiation. We have analyzed the methylation status of a set of miRNAs in a panel of neuroblastoma cell lines and identified a subset of hypermethylated and down-regulated miRNAs (miRNA 34b-3p, miRNA 34b-5p, miRNA34c-5p, and miRNA 124-2-3p) involved in the regulation of cell cycle, apoptosis and in the control of MYCN expression. These miRNAs share, in part, some of the targets whose expression is inversely correlated to the methylation and expression of the corresponding miRNA. To simulate the effect of the demethylation of miRNAs, we transfected the corresponding miRNA-mimics in the same cell lines and observed the down-regulation of a set of their target genes as well as the partial block of the cell cycle and the activation of the apoptotic pathway. The epigenetic alterations of miRNAs described in the present study were found also in a subset of patients at high risk of progression. Our data disclosed a complex network of interactions between epigenetically altered miRNAs and target genes, that could interfere at multiple levels in the control of cell homeostasis.

In a retrospective international study, circulating miR-148b and let-7b were found to be serum markers for detecting primary IgA nephropathy.

Immunoglobulin A nephropathy (IgAN) is a worldwide disease characterized by the presence of galactose-deficient IgA1 deposits in the glomerular mesangium. A kidney biopsy for diagnosis is required. Here, we measured two miRNAs (let-7b and miR-148b), previously identified as regulators of the O-glycosylation process of IgA1, in serum samples from patients with IgAN and healthy blood donors (controls) recruited in an international multicenter study. Two predictive models, based on these miRNAs, were developed and the diagnostic accuracy of the combined biomarkers was assessed by the area under the receiver operating characteristic (ROC) curve (AUC) carried out in three steps. In a training study, the combined miRNAs were able to discriminate between 100 patients with IgAN and 119 controls (AUC, 0.82). A validation study confirmed the model in an independent cohort of 145 patients with IgAN and 64 controls (AUC, 0.78). Finally, in a test study, the combined biomarkers were able to discriminate patients with IgAN from 105 patients affected by other forms of primary glomerulonephritis, supporting the specificity (AUC, 0.76). Using the same study design, we also performed two subgroup analyses (one for Caucasians and one for East Asians) and found that race-specific models were the best fit to distinguish IgAN patients from controls. Thus, serum levels of the combined miRNA biomarker, let-7b and miR-148b, appears to be a novel, reliable, and noninvasive test to predict the probability of having IgAN.

Aberrantly methylated DNA regions lead to low activation of CD4+ T-cells in IgA nephropathy.

IgAN (IgA nephropathy) is the most common form of primary glomerulonephritis worldwide and has a strong genetic component. In this setting, DNA methylation could also be an important factor influencing this disease. We performed a genome-wide screening for DNA methylation in CD4(+) T-cells from IgAN patients and found three regions aberrantly methylated influencing genes involved in the response and proliferation of CD4(+) T-cells. Two hypomethylated regions codified genes involved in TCR (T-cell receptor) signalling, TRIM27 (tripartite motif-containing 27) and DUSP3 (dual-specificity phosphatase 3), and an hypermethylated region included the VTRNA2-1 (vault RNA 2-1) non-coding RNA, also known as miR-886 precursor. We showed that the aberrant methylation influences the expression of these genes in IgAN patients. Moreover, we demonstrated that the hypermethylation of the miR-886 precursor led to a decreased CD4(+) T-cell proliferation following TCR stimulation and to the overexpression of TGFß (transforming growth factor ß). Finally, we found a Th1/Th2 imbalance in IgAN patients. The IL (interleukin)-2/IL-5 ratio was notably higher in IgAN patients and clearly indicated a Th1 shift. In conclusion, we identified for the first time some specific DNA regions abnormally methylated in IgAN patients that led to the reduced TCR signal strength of the CD4(+) T-cells and to their anomalous response and activation that could explain the T-helper cell imbalance. The present study reveals new molecular mechanisms underlying the abnormal CD4(+) T-cell response in IgAN patients.