OPA1 mutation affects autophagy and triggers senescence in autosomal dominant optic atrophy plus fibroblasts.

In several cases of mitochondrial diseases, the underlying genetic and bioenergetic causes of reduced oxidative phosphorylation (OxPhos) in mitochondrial dysfunction are well understood. However, there is still limited knowledge about the specific cellular outcomes and factors involved for each gene and mutation, which contributes to the lack of effective treatments for these disorders. This study focused on fibroblasts from a patient with Autosomal Dominant Optic Atrophy (ADOA) plus syndrome harboring a mutation in the Optic Atrophy 1 (OPA1) gene. By combining functional and transcriptomic approaches, we investigated the mitochondrial function and identified cellular phenotypes associated with the disease. Our findings revealed that fibroblasts with the OPA1 mutation exhibited a disrupted mitochondrial network and function, leading to altered mitochondrial dynamics and reduced autophagic response. Additionally, we observed a premature senescence phenotype in these cells, suggesting a previously unexplored role of the OPA1 gene in inducing senescence in ADOA plus patients. This study provides novel insights into the mechanisms underlying mitochondrial dysfunction in ADOA plus and highlights the potential importance of senescence in disease progression.

Mitofusin 2 mutation drives cell proliferation in Charcot-Marie-Tooth 2A fibroblasts.

Dominant mutations in ubiquitously expressed mitofusin 2 gene (MFN2) cause Charcot-Marie-Tooth type 2A (CMT2A; OMIM 609260), an inherited sensory-motor neuropathy that affects peripheral nerve axons. Mitofusin 2 protein has been found to take part in mitochondrial fusion, mitochondria-endoplasmic reticulum tethering, mitochondrial trafficking along axons, mitochondrial quality control and various types of cancer, in which MFN2 has been indicated as a tumor suppressor gene. Discordant data on the mitochondrial altered phenotypes in patient-derived fibroblasts harboring MFN2 mutations and in animal models have been reported. We addressed some of these issues by focusing on mitochondria behavior during autophagy and mitophagy in fibroblasts derived from a CMT2AMFN2 patient with an MFN2650G > T/C217F mutation in the GTPase domain. This study investigated mitochondrial dynamics, respiratory capacity and autophagy/mitophagy, to tackle the multifaceted MFN2 contribution to CMT2A pathogenesis. We found that MFN2 mutated fibroblasts showed impairment of mitochondrial morphology, bioenergetics capacity, and impairment of the early stages of autophagy, but not mitophagy. Unexpectedly, transcriptomic analysis of mutated fibroblasts highlighted marked differentially expressed pathways related to cell population proliferation and extracellular matrix organization. We consistently found the activation of mTORC2/AKT signaling and accelerated proliferation in the CMT2AMFN2 fibroblasts. In conclusion, our evidence indicates that MFN2 mutation can positively drive cell proliferation in CMT2AMFN2 fibroblasts.

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 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.

Altered monocyte expression and expansion of non-classical monocyte subset in IgA nephropathy patients.

BACKGROUND: The main defect of immunoglobulin A nephropathy (IgAN) lies within the immune system and in peripheral blood mononuclear cells rather than in the kidney. Previously, we found an altered gene expression in monocytes compared with B and T cells isolated from IgAN patients; thus, our aim here has been to study this subset at a genome-wide and functional level. METHODS: A total of 39 IgAN patients and 37 healthy blood donors (HBDs) were included in this study, and microarray technology was used to evaluate global gene expression differences in monocytes isolated from IgAN patients and HBDs. Aberrantly expressed genes and pathways were then validated on an independent set of IgAN patients with RT-PCR western blot and flow cytometric analysis. RESULTS: Gene expression differences in monocytes from IgAN patients and HBDs primarily involved apoptosis signalling, mitochondrial dysfunction, tnfr2/1 and death receptor signalling. Both the extrinsic and intrinsic apoptotic pathways seem to be implicated; in particular, the protein levels of NDUFS3 and TNFRSF1A were upregulated thus confirming the altered mitochondrial and death receptor homeostasis. Furthermore, the basal intracellular protein levels of TNF in monocytes were lower in IgAN patients compared with HBDs. We validated at protein level an enhanced apoptotic phenotype and a different subset distribution in monocytes from IgAN patients. We found that the non-classical monocyte subset (CD14(+)CD16(+)) was significantly expanded in all IgAN patients tested even though the total monocyte count remained unchanged. CONCLUSIONS: Our findings demonstrate, for the first time, an aberrant modulation of the mitochondrial respiratory system in monocytes isolated from IgAN patients. Furthermore, the aberrant expansion of the (CD14(+)CD16(+)) subset could explain the enhanced apoptotic phenotype seen in these cells thus revealing their potential role in the pathogenesis of IgAN.

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.

Kidney and urine cell transcriptomics in IgA nephropathy and lupus nephritis: a narrative review.

This narrative review sheds light on the use of transcriptomics in the analysis of kidney biopsies and urinary cell samples from patients with immunoglobulin A nephropathy or lupus nephritis. The conventional methods of examining kidney biopsy through light microscopy, immunofluorescence and electron microscopy provide valuable clinical information for diagnosis and prognosis but have some limitations that transcriptomics can address. Some recent studies have reported that kidney transcriptomics has uncovered new molecular biomarkers implicated in the inflammatory process induced by the deposition of circulating immune complexes in the investigated kidney diseases. In addition, transcriptomics applied to urinary cells mirrors the inflammatory process that occurs in the kidney. This means that we can study urinary cell transcriptomics in clinical practice to diagnose the stage of the inflammatory process. Furthermore, the transcriptomics of urinary cells can be used to make therapy decisions during patient follow-up to avoid the stress of a second kidney biopsy. The studies analyzed in this review have a significant limitation. Biomarkers have been identified in small cohorts of patients but none of them has been validated in independent external cohorts. Further prospective studies in large cohorts of patients are necessary for accurate and complete validation. Only after that can these biomarkers be widely used in clinical practice.

Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A and microvesicle-shuttled decorin.

Acute kidney injury (AKI) is emerging as a worldwide public health problem. Recent studies have focused on the possibility of using human adult renal stem/progenitor cells (ARPCs) to improve the repair of AKI. Here we studied the influence of ARPCs on the healing of cisplatin-injured renal proximal tubular epithelial cells. Tubular, but not glomerular, ARPCs provided a protective effect promoting proliferation of surviving tubular cells and inhibiting cisplatin-induced apoptosis. The recovery effect was specific to tubular ARPCs, occurred only after damage sensing, and was completely cancelled by TLR2 blockade on tubular ARPCs. Moreover, tubular, but not glomerular, ARPCs were resistant to the apoptotic effect of cisplatin. Tubular ARPCs operate mainly through the engagement of TLR2, the secretion of inhibin-A protein, and microvesicle-shuttled decorin, inhibin-A, and cyclin D1 mRNAs. These factors worked synergistically and were essential to the repair process. The involvement of tubular ARPC-secreted inhibin-A and decorin mRNA in the pathophysiology of AKI was also confirmed in transplant patients affected by delayed graft function. Hence, identification of this TLR2-driven recovery mechanism may shed light on new therapeutic strategies to promote the recovery capacity of the kidney in acute tubular damage. Use of these components, derived from ARPCs, avoids injecting stem cells.

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, ß1,3-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.

Dysregulation of testis mRNA expression levels in hatchery-produced vs wild greater amberjack Seriola dumerili.

Reproductive dysfunctions have been recently documented in male greater amberjack Seriola dumerili caught from the wild and reared in captivity. In the present study, we compared testis transcriptome in wild fish (WILD), hatchery-produced fish with apparently normal spermatogenesis (Normal Farmed; NormalF) and hatchery-produced fish with evident reproductive dysfunction (Dysfunctional Farmed; DysF). Gene expression analysis identified 2157, 1985 and 74 differentially expressed genes (DEGs) in DysF vs WILD, NormalF vs DysF and NormalF vs WILD comparisons, respectively. In DysF, a dysregulation of several interconnected biological processes, including cell assembly, steroidogenesis and apoptosis was found. Gene enrichment of progesterone-mediated oocyte maturation, oocyte meiosis and cell cycle pathways were identified in the DysF vs NormalF comparison. Most of the DEGs involved in the enriched pathways were downregulated in DysF. The comparison of NormalF vs WILD showed that most of the DEGs were downregulated in NormalF, including a gene that encodes for a regulatory protein with a protective role in apoptosis regulation (ptpn6), indicating that spermatogenesis was dysfunctional also in the apparently "normal" hatchery-produced fish. Hence, rearing of male greater amberjack in captivity, from eggs produced by captive breeders, did not prevent the appearance of reproductive dysfunctions, and these dysfunctions involved several biological processes and metabolic pathways.

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.

Racial heterogeneity of IgA1 hinge-region O-glycoforms in patients with IgA nephropathy.

Galactose (Gal)-deficient IgA1 (Gd-IgA1) is involved in IgA nephropathy (IgAN) pathogenesis. To reflect racial differences in clinical characteristics, we assessed disease- and race-specific heterogeneity in the O-glycosylation of the IgA1 hinge region (HR). We determined serum Gd-IgA1 levels in Caucasians (healthy controls [HCs], n = 31; IgAN patients, n = 63) and Asians (HCs, n = 20; IgAN patients, n = 60) and analyzed profiles of serum IgA1 HR O-glycoforms. Elevated serum Gd-IgA1 levels and reduced number of Gal residues per HR were observed in Caucasians. Reduced number of N-acetylgalactosamine (GalNAc) residues per HR and elevated relative abundance of IgA1 with three HR O-glycans were common features in IgAN patients; these features were associated with elevated blood pressure and reduced renal function. We speculate that the mechanisms underlying the reduced GalNAc content in IgA1 HR may be relevant to IgAN pathogenesis.

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.

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.

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.