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Novel functional changes during podocyte differentiation: increase of oxidative resistance and H-ferritin expression.

ABSTRACT: Podocytes are highly specialized, arborized epithelial cells covering the outer surface of the glomerular tuft in the kidney. Terminally differentiated podocytes are unable to go through cell division and hereby they are lacking a key property for regeneration after a toxic injury. Podocytes are long-lived cells but, to date, little is known about the mechanisms that support their stress resistance. Our aim was to investigate whether the well-known morphological changes during podocyte differentiation are accompanied by changes in oxidative resistance in a manner that could support their long-term survival. We used a conditionally immortalized human podocyte cell line to study the morphological and functional changes during differentiation. We followed the differentiation process for 14 days by time-lapse microscopy. During this period nondifferentiated podocytes gradually transformed into large, nonproliferating, frequently multinucleated cells, with enlarged nuclei and opened chromatin structure. We observed that differentiated podocytes were highly resistant to oxidants such as H2O2 and heme when applied separately or in combination, whereas undifferentiated cells were prone to such challenges. Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression. Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.

SUBMITTER: Banyai E

PROVIDER: S-EPMC4109136 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Novel functional changes during podocyte differentiation: increase of oxidative resistance and H-ferritin expression.

Bányai Emese E Balogh Enikő E Fagyas Miklós M Arosio Paolo P Hendrik Zoltán Z Király Gábor G Nagy Gábor G Tánczos Bence B Pócsi István I Balla György G Balla József J Bánfalvi Gáspár G Jeney Viktória V

Oxidative medicine and cellular longevity 20140706

Podocytes are highly specialized, arborized epithelial cells covering the outer surface of the glomerular tuft in the kidney. Terminally differentiated podocytes are unable to go through cell division and hereby they are lacking a key property for regeneration after a toxic injury. Podocytes are long-lived cells but, to date, little is known about the mechanisms that support their stress resistance. Our aim was to investigate whether the well-known morphological changes during podocyte different ...[more]

PMID: 25097723

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Project description:Glomerular podocytes are highly differentiated kidney cells that constitute one of the key components responsible for removing toxins and metabolic waste while preserving nutrients in the bloodstream in the renal glomerulus. In addition, podocytes contribute significantly to the formation of the glomerular basement membrane and the integrity of the vascular endothelium. Thus, podocyte injury and/or loss of podocytes results in impaired blood filtration and causes many common renal diseases characterized by severe proteinuria (protein in the urine) and hypoalbuminemia (low levels of blood albumin). Small lipopholic molecules such as steroids, fatty acids, prostaglandins and vitamin metabolites control many aspects of animal development, cell proliferation and differentiation, and homeostasis through binding to their intracellular receptors. Upon binding to their cognate ligands, these nuclear receptors (NRs) are capable of turning on or off of an array of gene networks. By doing so, they regulate a whole spectrum of cellular activities. The ability of small molecule hormones to regulate NR activity makes them excellent pharmaceutical targets. Clinical evidence and animal studies have implicated several NRs in contributing to podocyte development and disease. Recent studies from animals and cultured human or mouse podocytes indicate that synthetic hormones including estradiol, glucocorticoid, retinoid, pioglitazone, vitamin D3 and WY-14643 protect or rescue podocytes from experimental injury. Post treatment of injured podocytes with ligands for the above-mentioned NRs restores cytoskeletal architecture of the podocytes. Nonetheless, the mechanisms underlying the ability of these hormones in protecting podocytes remain largely unexplored. This is partly due to our limited knowledge of the target genes controlled by these hormones. In order to elucidate the mechanisms by which Dex, VD3 and ATRA elicit their renoprotective activity, we initiate a gene expression profiling study to identify the target genes of these hormones in cultured human podocytes. The temperature-sensitive cultured human podocytes (HPCs) were maintained in culture media of RPMI supplemented with 10% FBS, 1% antibiotics and the Insulin-Transferrin-Selenium at the permissive (undifferentiated) temperature of 33 Celsius degree. The 70%~80% confluent HPCs were induced to in vitro differentiate by culturing at 37 Celsius degree for 2 days then with incubation of vehicle (equal volume of DMSO), 100 nM all-trans retinoic acid (ATRA), 100 nM dexamethasone (Dex), or 100 nM vitamin D3 (VD3) treatment for 3 more days. The undifferentiated HPCs with vehicle (equal volume of DMSO) treatment was included as a control. The total RNAs of these cells were isolated using USB prepEase RNA spin kit following the manufacturer's instructions. Total 1 microgram RNA at 100 nanogram per microliter for each sample were sent for a single-channel gene expression microarray studies using HumanRef-8 chip (V3_0_R3_11282963) on Illumina BeadStation platform. The RNA were converted to aRNA and appropriately labelled using MessageAmp Biotin aRNA Amplification Kit (Ambion) following the manufacturer's instructions. The raw data were collected by scanning the chip on Illumina BeadStation. Background subtracted raw data were exported and the following analysis was done in R/Bioconductor environment. Thus, we were able to compare the gene expression profiles between differentiated and undifferentiated HPCs with or without hormone treatment.

Project description:Objective: It has been demonstrated that the transcription factors TAZ (transcriptional coactivator with PDZ-binding motif), paired box gene 8 (PAX8), and NK2 homeobox 1 (NKX2-1) are coexpressed in the nucleus of thyroid cells. Furthermore, TAZ is known to enhance the transcriptional activity of PAX8 and NKX2-1 as well as the key thyroid-specific gene, thyroglobulin (TG), suggesting a critical role for TAZ in the control of thyroid cell speciation. We previously reported that the small molecule ethacridine, identified as a TAZ activator, was able to induce thyroid-specific transcription in endodermal cells differentiated from human embryonic stem (hES) cells using activin A. Since transcription factors are epigenetically regulated in cell differentiation, we investigated the epigenetic changes in the promoter regions of these key transcription factors during in vitro differentiation of hES cells into thyrocytes. Methods: We initially profiled chromatin accessibility using the technique of Assay for Transposase Accessible Chromatin sequencing (ATAC-seq), and then examined DNA methylation and histone acetylation in the promoter regions of the three selected thyroid transcription factors and the thyroid-specific genes during hES cell differentiation. Results: ATAC-seq analysis showed enriched chromatin accessibility of TAZ, NKX2-1, and PAX8 after exposure to activin A and ethacridine. There were no methylation changes found in the NKX2-1, PAX8, and TAZ promoters by bisulfite sequencing. In contrast, acetylation of histone H4, specifically acetylation of lysine 16, was observed in each of the promoters when measured by chromatin immunoprecipitation polymerase chain reaction assays, which correlated with the activity and expression of NKX2-1 and PAX8 as well as sodium/iodide symporter, thyroid stimulating hormone receptor, and TG genes. Conclusions: These results indicate that ethacridine treatment of activin A-derived endodermal hES cells leads to enhanced chromatin accessibility, which, in turn, allows histone H4 acetylation in the regulation of active genes for speciation of thyroid follicular cells from hES cells.

Project description:Ferritin is the major intracellular iron storage protein that sequesters excess free iron to minimize generation of iron-catalysed reactive oxygen species. We previously demonstrated that expression of ferritin heavy chain (ferritin H) was induced by pro-oxidants, which is a part of cellular antioxidant response to protect cells from oxidative damage. In this study, we have identified that the antioxidant/electrophile response element (ARE) located 4.5 kb upstream to the human ferritin H transcription initiation site is responsible for the oxidant response. The human ferritin H ARE comprises two copies of bidirectional AP1 motifs. Mutations in each AP1 motif significantly impaired protein binding and the function of the ARE, indicating that both of the AP1 motifs are required for pro-oxidant-mediated activation of the ferritin H gene. We identified that JunD, an AP1 family basic-leucine zipper (bZip) transcription factor, is one of the ferritin H ARE binding proteins and activates ferritin H transcription in HepG2 hepatocarcinoma cells. Gel retardation assay demonstrated that H2O2 (hydrogen peroxide) or t-BHQ (tert-butylhydroquinone) treatment increased total protein binding as well as JunD binding to the ferritin H ARE. Chromatin immunoprecipitation assay showed that H2O2 treatment induced JunD binding to the ferritin H ARE. Both H2O2 and t-BHQ induced phosphorylation of JunD at Ser-100, an activated form of JunD. Furthermore, overexpression of JunD induced endogenous ferritin H protein synthesis. Since JunD has recently been demonstrated to protect cells from several stress stimuli including oxidative stress, these results suggest that, in addition to NFE2-related factor 2 (Nrf2) as a major ARE regulatory protein, JunD is another ARE regulatory protein for transcriptional activation of the human ferritin H gene and probably other antioxidant genes containing the conserved ARE sequences by which JunD may confer cytoprotection during oxidative stress.

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Novel functional changes during podocyte differentiation: increase of oxidative resistance and H-ferritin expression.

Publications

Novel functional changes during podocyte differentiation: increase of oxidative resistance and H-ferritin expression.

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