Grazia Serino

Abnormal miR-148b Expression Promotes Aberrant Glycosylation of IgA1 in IgA Nephropathy

Aberrant O-glycosylation in the hinge region of IgA1 characterizes IgA nephropathy. The mechanisms underlying this abnormal glycosylation are not well understood, but reduced expression of the enzyme core 1, β1,3-galactosyltransferase 1 (C1GALT1) may contribute. In this study, high-throughput microRNA (miRNA) profiling identified 37 miRNAs differentially expressed in PBMCs of patients with IgA nephropathy compared with healthy persons. Among them, we observed upregulation of miR-148b, which potentially targets C1GALT1. Patients with IgA nephropathy exhibited lower C1GALT1 expression, which negatively correlated with miR-148b expression. Transfection of PBMCs from healthy persons with a miR-148b mimic reduced endogenous C1GALT1 mRNA levels threefold. Conversely, loss of miR-148b function in PBMCs of patients with IgA nephropathy increased C1GALT1 mRNA and protein levels to those observed in healthy persons. Moreover, we found that upregulation of miR-148b directly correlated with levels of galactose-deficient IgA1. In vitro, we used an IgA1-producing cell line to confirm that miR-148b modulates IgA1 O-glycosylation and the levels of secreted galactose-deficient IgA1. Taken together, these data suggest a role for miRNAs in the pathogenesis of IgA nephropathy. Abnormal expression of miR-148b may explain the aberrant glycosylation of IgA1, providing a potential pharmacologic target for IgA nephropathy.

MicroRNAs in kidney diseases: new promising biomarkers for diagnosis and monitoring

A series of microRNAs (miRNAs) have a critical role in many cellular and physiological activities such as cell cycle, growth, proliferation, apoptosis and metabolism. miRNAs are also important in the maintenance of renal homeostasis and kidney diseases. In vitro and in vivo animal models have shown a critical role of miRNAs in the development of diabetic nephropathy (DN) and in the progression of renal fibrosis. Specific miRNAs in renal tissue and peripheral blood mononuclear cells (PBMCs) are up and downregulated in different kidney diseases. They represent new potential biomarkers for diagnosis and targeted therapy. In addition, urinary miRNAs may be considered non-invasive biomarkers for monitoring the progression of renal damage. The activity of miRNAs can be modified by different approaches such as the use of antisense oligonucleotide inhibitors (antagomirs), tandem miRNA-binding site repeats manufactured by Decoy or Sponge technologies and miRNA mimics. The use of miRNA blockers or antagonists as therapeutic agents is very attractive but new information will be necessary considering their role in other systems.

Altered modulation of WNT-beta-catenin and PI3K/Akt pathways in IgA nephropathy.

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. The basic defect lies within the IgA immune system and in peripheral blood leukocytes, rather than local kidney abnormalities. To define the intracellular mechanisms leading to the disease, we conducted a microarray study to identify genes and pathways differentially modulated in peripheral blood leukocytes isolated from 12 IgAN patients and 8 healthy controls. The genes whose expression discriminated between the IgAN patients and controls were primarily involved in canonical WNT-beta-catenin and PI3K/Akt pathways. We also tested peripheral blood mononuclear cells and their subpopulations isolated from an independent group of IgAN patients and healthy controls. There were low protein levels of inversin and PTEN, key regulators of WNT-beta-catenin and PI3K/Akt, in IgAN patients, suggesting hyperactivation of these pathways. Also, there were increased phospho-Akt protein levels and nuclear beta-catenin accumulation with an enhanced peripheral blood mononuclear cell proliferation rate. Subpopulation analysis uncovered a major irregularity of WNT signaling in monocytes. Hence, hyperactivation of these pathways may provide insight into mechanisms contributing to the pathogenesis of IgAN.

Altered modulation of WNT–β-catenin and PI3K/Akt pathways in IgA nephropathy

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. The basic defect lies within the IgA immune system and in peripheral blood leukocytes, rather than local kidney abnormalities. To define the intracellular mechanisms leading to the disease, we conducted a microarray study to identify genes and pathways differentially modulated in peripheral blood leukocytes isolated from 12 IgAN patients and 8 healthy controls. The genes whose expression discriminated between the IgAN patients and controls were primarily involved in canonical WNT-beta-catenin and PI3K/Akt pathways. We also tested peripheral blood mononuclear cells and their subpopulations isolated from an independent group of IgAN patients and healthy controls. There were low protein levels of inversin and PTEN, key regulators of WNT-beta-catenin and PI3K/Akt, in IgAN patients, suggesting hyperactivation of these pathways. Also, there were increased phospho-Akt protein levels and nuclear beta-catenin accumulation with an enhanced peripheral blood mononuclear cell proliferation rate. Subpopulation analysis uncovered a major irregularity of WNT signaling in monocytes. Hence, hyperactivation of these pathways may provide insight into mechanisms contributing to the pathogenesis of IgAN.

miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity.

Role of let-7b in the regulation of N-acetylgalactosaminyltransferase 2 in IgA nephropathy

BACKGROUND IgA nephropathy (IgAN) is characterized by aberrant O-glycosylation in the hinge region of IgA1. The early step in O-glycan formation is the attachment of N-acetylgalactosamine (GalNAc) to the serine/threonine of the hinge region; the process is catalysed by UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). In our previous work, the microarray analysis on peripheral blood mononuclear cells (PBMCs) identified an upregulated miRNA called let-7b. METHODS To study the molecular mechanisms in which let-7b was involved, we performed a bioinformatic analysis to predict their target genes. To validate biologically let-7b targets, we performed transient transfection experiments ex vivo using PBMCs from an independent group of IgAN patients and healthy blood donors (HBDs). RESULTS Bioinformatic analysis revealed that GALNT2 is the potential target of let-7b. We found this miRNA significantly upregulated in PBMCs of IgAN patients compared with HBDs. Then, we demonstrated in ex-vivo experiments that let-7b decreased GALNT2 levels in PBMCs of IgAN patients, whereas the loss of let-7b function in PBMCs of HBDs led to an increase of GALNT2 mRNA and its protein level. Finally, we found that upregulation of let-7b occurred also in B-lymphocytes from IgAN patients. CONCLUSIONS Our results give novel additional information on the abnormal O-glycosylation process of IgA1 in IgAN patients. This study provides evidence for another important miRNA-based regulatory mechanism of the O-glycosylation process in which the deregulated expression of let-7b is associated with altered expression of GALNT2. This finding could be taken into consideration for new therapeutic approaches in IgAN because other serum glycosylated proteins do not display abnormal glycosylation.

Activated innate immunity and the involvement of CX3CR1–fractalkine in promoting hematuria in patients with IgA nephropathy

A hallmark of immunoglobulin A nephropathy (IgAN) is episodes of gross hematuria coinciding with mucosal infections that can represent the disease-triggering event. Here we performed a whole genomic screen of IgAN patients during gross hematuria to clarify the link between mucosal antigens and glomerular hematuria. Modulated genes showed a clear involvement of the intracellular interferon signaling, antigen-presenting pathway, and the immunoproteasome. The mRNA and protein level of the chemokine receptor characterizing cytotoxic effector lymphocytes, CX3CR1, was upregulated. In vitro antigenic stimulation of peripheral blood mononuclear cells from IgAN patients, healthy blood donors, and other nephropathies with microscopic hematuria showed that only in IgAN patients was CX3CR1 enhanced in a dose-dependent manner. A significantly higher amount of glomerular and urinary fractalkine, the only ligand of CX3CR1, was also found in IgAN patients with recurrent episodes of gross hematuria compared with other patients with microscopic or no hematuria. This suggests a predisposition for cytotoxic cell extravasation only in patients with recurrent gross hematuria. Thus, we found a defect in antigen handling in peripheral blood mononuclear cells of IgAN patients with a specific increase of CX3CR1. This constitutive upregulation of glomerular and urinary fractalkine suggests an involvement of the CX3CR1-fractalkine axis in the exacerbation of gross hematuria.

microRNAs in glomerular diseases from pathophysiology to potential treatment target

miRNAs are regulators of gene expression in diverse biological and pathological courses in life. Their discovery may be considered one of the most important steps in the story of modern biology. miRNAs are packed within exosomes and released by cells for cellular communications; they are present in bodily fluids. Their study opens the way for understanding the pathogenetic mechanisms of many diseases; furthermore, as potential candidate biomarkers, they can be measured in bodily fluids for non-invasive monitoring of disease outcomes. The present review highlights recent advances in the role of miRNAs in the pathogenesis of primary and secondary glomerulonephritides such as IgA nephropathy, focal segmental glomerular sclerosis, lupus nephritis and diabetic nephropathy. The identification of reciprocal expression of miRNAs and their target genes provides the molecular basis for additional information on the pathogenetic mechanisms of kidney diseases. Finally, recent findings demonstrate that miRNAs can be considered as potential targets for novel drugs.

Dendritic Cells Modulate Iron Homeostasis and Inflammatory Abilities Following Quercetin Exposure

BACKGROUND Fruits and vegetables are rich in plant polyphenols, whose consumption is encouraged in healthy dietary regimes due to their antioxidants and anti-inflammatory effects. These organic molecules exhibit numerous properties including phylochelation; the ability to complex metal ions, including highly reactive iron. Among polyphenols, we focused our attention on quercetin that previously demonstrated its ability to reduce dendritic cells (DCs) inflammatory cytokine secretion and antigen presentation following LPS exposure. Dendritic cell inflammatory response is also associated with modulation of several iron metabolism related genes. OBJECTIVE To characterize the axis between quercetin exposure and iron extracellular transport that may explain polyphenol anti-inflammatory abilities. METHOD Bone marrow derived DCs were exposed to 25μM of quercetin on day 7 and treated with 1 μg/mL of LPS on day 8. The relation between quercetin exposure and the expression level of genes involved in iron homeostasis was addressed by qPCR. The axis between iron export and quercetin exposure was confirmed in vitro and in vivo using quercetin gavage and quercetin-enriched diet. RESULTS Here we demonstrate that DCs, exposed to quercetin, activate a pattern of genes that increase extracellular iron export, resulting in an overall decrease in the intracellular iron content and consequent diminished inflammatory abilities. This DCs phenotype is consistent with anti-inflammatory phenotype of the mucosal resident DCs, the ones most commonly exposed to polyphenols. CONCLUSIONS Iron balance is a crucial checkpoint for DCs inflammatory abilities. Quercetin-enriched nutritional regimes that favor DCs extracellular iron transport could reduce the incidence of chronic inflammatory syndromes.

Genome-wide scan identifies a copy number variable region at 3p21.1 that influences the TLR9 expression levels in IgA nephropathy patients

Immunoglobulin A nephropathy (IgAN) is a complex multifactorial disease characterized by genetic factors that influence the pathogenesis of the disease. In this context, an intriguing role could be ascribed to copy number variants (CNVs). We performed the whole-genome screening of CNVs in familial IgAN patients, their healthy relatives and healthy subjects (HSs). In the initial screening, we included 217 individuals consisting of 51 biopsy-proven familial IgAN cases and 166 healthy relatives. We identified 148 IgAN-specific aberrations, specifically 105 loss and 43 gain, using a new statistical approach that allowed us to identify aberrations that were concordant across multiple samples. Several CNVs overlapped with regions evidenced by previous genome-wide genetic studies. We focused our attention on a CNV located in chromosome 3, which contains the TLR9 gene and found that IgAN patients characterized by deteriorated renal function carried low copy number of this CNV. Moreover, the TLR9 gene expression was low and significantly correlated with the loss aberration. Conversely, IgAN patients with normal renal function had no aberration and the TLR9 mRNA was expressed at the same level as in HSs. We confirmed our data in another cohort of Greek subjects. In conclusion, here we performed the first genome-wide CNV study in IgAN identifying structural variants that could help the genetic dissection of this complex disease, and pointed out a loss aberration in the chromosome 3, which is responsible for the downregulation of TLR9 expression that, in turn, could contribute to the deterioration of the renal function in IgAN patients.