ABSTRACT
<p><b>OBJECTIVE</b>To study the effect of arctiin on mouse podocyte epithelial-mesenchymal transition (EMT) induced by advanced oxidation protein products (AOPP).</p><p><b>METHODS</b>Mouse podocytes were stimulated by 200 µg/ml AOPP for 24 h in the presence of 50, 100, 200, and 400 µmol/L arctiin. The expressions of α-smooth muscle actin, Grp78 and CHOP were detected using Western blotting.</p><p><b>RESULT</b>The expressions of α-SMA, Grp78 and CHOP were inhibited by arctiin, showing a dose-dependent effect within a given range of arctiin concentration.</p><p><b>CONCLUSION</b>AOPP causes endoplasmic reticulum stress to induce EMT of mouse podocytes, and arctiin can decrease EMT by alleviating the stress. This finding sheds light on a new scope of research of renal fibrosis.</p>
Subject(s)
Animals , Mice , Actins , Metabolism , Advanced Oxidation Protein Products , Cell Line , Endoplasmic Reticulum Stress , Epithelial-Mesenchymal Transition , Furans , Pharmacology , Glucosides , Pharmacology , Heat-Shock Proteins , Metabolism , Podocytes , Metabolism , Pathology , Transcription Factor CHOP , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To detect the effect of connective tissue growth factor (CTGF) on podocalyxin expression in mouse podocytes exposed to high glucose in vitro and explore the possible pathway involved.</p><p><b>METHODS</b>The expression vector carrying a small interfering RNA (siRNA) targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose (normal control), 4.5 g/L glucose (high glucose group), 1 g/L glucose + 3.5 g/L mannitol (iso-osmolar control group). The changes in the protein expression levels of podocalyxin, CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting.</p><p><b>RESULTS</b>High glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes (P<0.05). Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure, and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes.</p><p><b>CONCLUSION</b>CTGF is an important mediator of high glucose-induced podocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury, suggesting its potential value in treatment of diabetic nephropathy.</p>
Subject(s)
Animals , Mice , Cells, Cultured , Connective Tissue Growth Factor , Metabolism , Diabetic Nephropathies , Glucose , MAP Kinase Signaling System , Podocytes , Cell Biology , Metabolism , RNA, Small Interfering , Genetics , Sialoglycoproteins , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the effect of transfection with small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on human tubular epithelial hypertrophy induced by high glucose.</p><p><b>METHODS</b>HK-2 cells were cultured in DMEM/F12 medium containing 1 g/L glucose (normal control group), 4.5 g/L glucose (high glucose group), or 1 g/L glucose+3.5 g/L mannitol (iso-osmolar control group). The cells were transfected with pGenesil-1, pGenesil/neg, or pGenesil/siRNA-CTGF and then cultured in DMEM/F12 medium containing 4.5 g/L glucose as the high glucose+blank control group, high glucose+negative control group and high glucose+interference group, respectively. After cell culture for 24, 48 and 96 h, the cells were collected to detect the mRNA and protein levels of CTGF by real-time PCR and Western blotting, respectively. The proliferative activities of the cells were evaluated with MTT assay, and the total cellular protein contents were determined with Bradford method. Flow cytometry was employed to analyzed the cell cycle changes.</p><p><b>RESULTS</b>High-glucose significantly up-regulated the CTGF mRNA and protein levels in HK-2 cells. The cell proliferation was inhibited after high-glucose exposure with increased cell percentage in G1 phase and total cellular protein content suggesting cellular hypertrophy. Transfection with siRNA targeting CTGF significantly inhibited high glucose-induced up-regulation of CTGF mRNA and protein and promoted the cell proliferation, resulting also increased cells in S phase and lowered total cellular protein contents.</p><p><b>CONCLUSION</b>CTGF is an important mediator of high glucose-induced tubular epithelial hypertrophy, and transfection with siRNA targeting CTGF can alleviate the hypertrophy, suggesting the potential value of CTGF-targeted treatment in the management of diabetic nephropathy.</p>
