Dataset Information

Bcl-2-modifying factor induces renal proximal tubular cell apoptosis in diabetic mice.

ABSTRACT: This study investigated the mechanisms underlying tubular apoptosis in diabetes by identifying proapoptotic genes that are differentially upregulated by reactive oxygen species in renal proximal tubular cells (RPTCs) in models of diabetes. Total RNAs isolated from renal proximal tubules (RPTs) of 20-week-old heterozygous db/m+, db/db, and db/db catalase (CAT)-transgenic (Tg) mice were used for DNA chip microarray analysis. Real-time quantitative PCR assays, immunohistochemistry, and mice rendered diabetic with streptozotocin were used to validate the proapoptotic gene expression in RPTs. Cultured rat RPTCs were used to confirm the apoptotic activity and regulation of proapoptotic gene expression. Additionally, studies in kidney tissues from patients with and without diabetes were used to confirm enhanced proapoptotic gene expression in RPTs. Bcl-2-modifying factor (Bmf) was differentially upregulated (P<0.01) in RPTs of db/db mice compared with db/m+ and db/db CAT-Tg mice and in RPTs of streptozotocin-induced diabetic mice in which insulin reversed this finding. In vitro, Bmf cDNA overexpression in rat RPTCs coimmunoprecipated with Bcl-2, enhanced caspase-3 activity, and promoted apoptosis. High glucose (25 mmol/L) induced Bmf mRNA expression in RPTCs, whereas rotenone, catalase, diphenylene iodinium, and apocynin decreased it. Knockdown of Bmf with small interfering RNA reduced high glucose-induced apoptosis in RPTCs. More important, enhanced Bmf expression was detected in RPTs of kidneys from patients with diabetes. These data demonstrate differential upregulation of Bmf in diabetic RPTs and suggest a potential role for Bmf in regulating RPTC apoptosis and tubular atrophy in diabetes.

SUBMITTER: Lau GJ

PROVIDER: S-EPMC3266424 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Bcl-2-modifying factor induces renal proximal tubular cell apoptosis in diabetic mice.

Lau Garnet J GJ Godin Nicolas N Maachi Hasna H Lo Chao-Sheng CS Wu Shyh-Jong SJ Zhu Jian-Xin JX Brezniceanu Marie-Luise ML Chénier Isabelle I Fragasso-Marquis Joelle J Lattouf Jean-Baptiste JB Ethier Jean J Filep Janos G JG Ingelfinger Julie R JR Nair Viji V Kretzler Matthias M Cohen Clemens D CD Zhang Shao-Ling SL Chan John S D JS

Diabetes 20111230 2

This study investigated the mechanisms underlying tubular apoptosis in diabetes by identifying proapoptotic genes that are differentially upregulated by reactive oxygen species in renal proximal tubular cells (RPTCs) in models of diabetes. Total RNAs isolated from renal proximal tubules (RPTs) of 20-week-old heterozygous db/m+, db/db, and db/db catalase (CAT)-transgenic (Tg) mice were used for DNA chip microarray analysis. Real-time quantitative PCR assays, immunohistochemistry, and mice rendere ...[more]

PMID: 22210314

Similar Datasets

Project description:BackgroundDiabetic kidney disease (DKD) has mainly been considered as a glomerular disease. Our previous study showed that the progression of DKD was highly correlated with the dysfunction of renal proximal tubular cells. Fermented Cordyceps sinensis (CS), a substitute for natural CS, is a prominent herb widely used in China, and has exhibited excellent efficacy on DKD. However, the underlying mechanisms remain poorly understood.MethodsThe database analysis was used to identify the main therapeutic targets and pathways of CS involved in DKD treatment. Next, the protective effects of fermented CS on high glucose (HG, 30 mM) induced HK-2 cell injury was validated through cell proliferation and apoptosis assay, including CCK-8, EdU and TUNEL. Finally, quantitative real‑time PCR (qRT-PCR) and western blotting were used to verify key target genes.ResultsOur results revealed that 9 main targets (RELA, JNK1, PTEN, VEGFA, EGF, ERK2, CASP3, AKT1, MMP9) were recognized as key therapeutic targets with excellent binding affinity screened by database analysis and molecular docking. The biological processes were identified by Gene Ontology (GO) enrichment, which appeared mainly involved in the positive regulation of cell proliferation as well as the negative regulation of apoptosis. The verification experiments in vitro revealed that fermented CS significantly attenuated the HG-induced cytotoxicity and apoptosis, and promoted the proliferation of HK-2 cells. Moreover, fermented CS significantly downregulated the expressions of Bax, Caspase-3, VEGFA, P-AKT and P-ERK, and upregulated the expression of PTEN compared with that of HG group.ConclusionOur results demonstrate that the fermented CS has nephroprotective effects significantly, which functions via promoting proliferation and inhibiting apoptosis of renal proximal tubular cells, likely by targeting Caspase-3, Bax, VEGFA and PTEN. Furthermore, AKT and ERK signaling pathway may be the critical mechanisms underlying the efficacy of fermented CS in DKD treatment.

Project description:BackgroundMelanoma-associated antigen H1 (MAGEH1) is a protein that belongs to melanoma-associated antigen (MAGE) superfamily. Growth arrest and DNA damage 45G (GADD45G) is a member of the DNA damage-inducible gene family which responds to environmental stresses. We have previously shown that GADD45G is a protein that promotes apoptosis of renal tubular cells in response to a nephrotoxic injury. In this study, we show evidence that MAGEH1 interacts with GADD45G and is involved in the induction of nephrotoxin-induced apoptosis of renal tubular cells.MethodsPrimary human renal tubular epithelial (HRE) cells and human kidney 2 (HK-2) cells were used in this study. To produce stable cell lines in which MAGEH1 expression was silenced, HRE cells were transduced with a lentiviral vector encoding a single guide RNA construct targeting the MAGEH1 gene. To knockdown GADD45G expression in HRE cells, a vector containing short hairpin RNA (shRNA) was used. We used short interfering RNAs (siRNA) to achieve transient silencing of genes in HK-2 cells. Recombinant adenoviruses were synthesized to overexpress MAGEH1 and GADD45G proteins. Human protein microarray was used to identify proteins that binds to GADD45G. Co-immunoprecipitation assays were then performed to confirm microarray results. Cell death was induced by cyclosporine A (CsA). Real-time quantitative PCR assay was used to evaluate gene expression levels. The degree of apoptosis and necrosis of cultured cells was evaluated by flow cytometry. Expression levels of caspases were examined using western blot analysis.ResultsWe found that GADD45G bound to one protein spotted in the protein microarray, which was subsequently identified as MAGEH1. We confirmed the interaction between GADD45G and MAGEH1 protein using the co-immunoprecipitation assay. MAGEH1 gene expression was not altered by CsA-induced cytotoxic injury, whereas GADD45G gene expression was increased significantly upon CsA treatment. MAGEH1 expression was significantly downregulated in GADD45G knockdown HRE stable cells suggesting that MAGEH1 expression may be dependent on GADD45G expression. CsA-induced apoptosis was significantly reduced in MAGEH1 knockdown HRE stable cells which led to an increased survival of these cells. Similar results were observed in GADD45G knockdown HRE stable cells. Accordingly, CsA-induced apoptosis was significantly decreased in MAGEH1 siRNA and GADD45G siRNA transfected HK-2 cells. CsA-induced activation of caspase-7 and caspase-9 was inhibited in MAGEH1 knockdown HRE stable cells, and similarly in GADD45G knockdown HRE stable cells.ConclusionsTo the best of our knowledge, this is the first study to show that MAGEH1 interacts with GADD45G and that MAGEH1 is involved in caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

Dataset Information

Bcl-2-modifying factor induces renal proximal tubular cell apoptosis in diabetic mice.

Publications

Bcl-2-modifying factor induces renal proximal tubular cell apoptosis in diabetic mice.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets