Unknown,Transcriptomics,Genomics,Proteomics

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Role of calcium/calmodulin-dependent kinase type IV in podocyte function in lupus nephritis

ABSTRACT: Kidney podocytes and their slit diaphragms contribute to prevent urinary protein loss. T cell from patients with systemic lupus erythematosus display increased expression of calcium/calmodulin kinase IV (CaMKIV). Here we evaluated the functional role of CaMKIV in lupus nephritis (LN) using kidney biopsy specimens and human podocyte cell line (AB8/13). We found that exposure of podocytes to IgG from LN patients resulted in entry of IgG into the cytoplasm. CaMKIV expression was found to be increased in podocytes of LN kidney biopsy specimens and exposure to IgG from LN patients. IgG entered podocytes using the FcRn receptor because when podocytes where treated with FcRn siRNA less IgG was found in the cytoplasm. The DNA microarray studies of podocytes exposed to LN IgG revealed that genes that are related to the activation of immune cells or podocyte damage were upregulated. These genes included CD86, CaMKIV, PTPN22, PDE5A, CD47 and MALT1. Interestingly, CD86 expression decreased after silencing CaMKIV in podocytes. Also, in situ hybridization experiments showed that the expression of CD86 was reduced in podocytes from MRL/lpr.camkivM-bM-^HM-^R/M-bM-^HM-^R mice. IgG from LN patients may enter podocytes through the FcRn and causes the upregulation of a distinct set of genes which may alter podocyte function. Upregulation of CaMKIV appears to precede that of genes known to be linked to podocyte damage such as CD86. These findings may indicate that inhibition of CaMKIV may prove of clinical use in patients with LN. IgG Purification Kits (Dojindo Molecular Technologies, Inc.) are used for isolation and purification of immunoglobulin G of healthy and normal individual according to the manufacturerM-bM-^@M-^Ys protocol. Flow through the column was used for non IgG binding samples. Cultured human podocytes with IgG purified from sera of normal individuals and LN patient for 24 hr were collected for RNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Kunihiro Ichinose

PROVIDER: E-GEOD-55768 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We recently revealed that myeloid master regulator PU.1 directly represses metallothionein (MT)-1G through its epigenetic activity, but the functions of MT-1G in myeloid differentiation remain unknown. To clarify this, we established MT-1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT-1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were attenuated in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, defensin-4, C-X3-C motif receptor 3, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT-1G disturbs the proper differentiation of myeloid cells. The present study provides evidence that expression analysis of MT-1G in acute promyelocytic leukemia patients may be a good prediction marker to estimate the efficacy of ATRA. Cell culture and generation of MT-1G-overexpressing cells: To generate MT-1G-overexpressing cells and their control cells, the MT-1G expression vector and its parental pcDNA 3.1/myc-His(-) version A vector (Invitrogen) were transfected using a CLB-Transfection device (Lonza, Basel, Switzerland). NB4 clones stably transfected with the vectors were isolated by limiting dilution and selection with 400 µg/ml of neomycin in RPMI (Gibco BRL, Rockville, MD) containing 10% heat-inactivated fetal bovine serum (HIFBS). Cells were cultured under 5% CO2 at 37°C in a humidified atmosphere. Microarray analyses: MT-1G-overexpressing (NB4MTOE) cells and their control cells were seeded at a density of 1×105 cells/ml and treated with 1 µM all-trans retinoic acid (ATRA). The cells were harvested after 72 h and total cellular RNA was isolated from control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells using an RNA Mini Purification Kit (Qiagen, Miami, FL) according to the manufacturer’s protocol. Aliquots containing 10 µg of RNA from each sample of control cells were mixed and used as controls. Similarly, 10 µg of RNA from each sample of NB4MTOE cells were mixed and used as NB4MTOE cells. The samples were subjected to microarray analyses using a CodeLink Human 54K Whole Genome Bioarray (Filgen, Nagoya, Japan).

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Role of calcium/calmodulin-dependent kinase type IV in podocyte function in lupus nephritis

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