1.Study of resveratrol suppressing TGF-beta1 induced transdifferentiation of podocytes.
Ru-Chun YANG ; Xiao-Ling ZHU ; Hua-Qin ZHANG ; Wei-Dong LI
Chinese Journal of Integrated Traditional and Western Medicine 2013;33(12):1677-1682
OBJECTIVETo explore the effect of resveratrol on transforming growth factor-beta1 (TGF-beta1) induced transdifferentiation of podocytes.
METHODSMouse podocytes in vitro cultured under differentiating conditions for 10 days were divided into the normal group, the model group, the high dose resveratrol group, and the low dose resveratrol group. The podocytes in the high and low dose resveratrol groups were intervened with 5 micromol/L and 2 micromol/L resveratrol respectively for 30 min. Those in the model group and the two resveratrol treated groups were continually incubated with 5 ng/mL TGF-beta1 for 72 h. Those in the normal group were routinely cultured. The protein expression of podocyte phenotypic protein molecules such as E-cadherin, P-cadherin, zonula occludens-1 (ZO-1), NEPH1, and alpha-smooth muscle-actin (alpha-SMA) were detected by immunocytochemistry, flow cytometry (FCM), and Western blot. A simple albumin influx assay was used to evaluate the filtration barrier function of podocyte monolayer.
RESULTSCompared with the normal control group, E-cadherin (+) percentage rate, the protein expression of P-cadherin, ZO-1, and NEPH1 significantly decreased in the model group (P < 0.05), but the expression of alpha-SMA and albumin permeability across podocyte monolayers increased significantly (P < 0.05). Compared with the model group, E-cadherin (+) percentage rate significantly increased (P < 0.05) and albumin permeability across podocyte monolayers decreased significantly (P < 0.05) in the high and low dose resveratrol groups. In the low dose resveratrol group, the expression of P-cadherin and NEPH1 significantly increased (P < 0.05). In the high dose resveratrol group, the expression of P-cadherin, ZO-1, and NEPH1 increased significantly, and the expression of alpha-SMA decreased significantly (P < 0.05). The correlations between resveratrol concentrations and the expression of E-cadherin (+), P-cadherin, and NEPH1 were significantly positive (r(E-cadherin (+)) = 0.772, r(P-cadherin) = 0.756, r(NEPH1) = 0.809, P < 0.05).
CONCLUSIONThe role of resveratrol in inhibiting TGF-beta1 induced phenotype abnormality might be an important mechanism for preserving the integrality of glomerular filtration barrier and decreasing proteinuria.
Animals ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Kidney Tubules ; cytology ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; Stilbenes ; pharmacology ; Transforming Growth Factor beta1 ; metabolism
2.Connective tissue growth factor mediates high glucose-induced down-regulation of podocalyxin expression in mouse podocytes.
Jun ZHANG ; Ping-hua LI ; Lei YANG ; Qing-sheng DU ; Ting-ting GUO ; Xun TANG
Journal of Southern Medical University 2011;31(5):839-843
OBJECTIVETo 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.
METHODSThe 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.
RESULTSHigh 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.
CONCLUSIONCTGF 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.
Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; metabolism ; Diabetic Nephropathies ; Glucose ; adverse effects ; MAP Kinase Signaling System ; drug effects ; Mice ; Podocytes ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Sialoglycoproteins ; metabolism
3.Effect of Wenyang Huoxue Lishul Recipe Containing Serum on Expression of Cathepsin L in Puromycin Aminonucleoside-induced Injury of Mouse Glomerular Podocytes.
Wen-wen QIU ; Jun YUAN ; Liu YANG
Chinese Journal of Integrated Traditional and Western Medicine 2016;36(5):602-607
OBJECTIVETo observe the effect of Wenyang Huoxue Lishui Recipe (WHLR) containing serum on the expression of cathepsin L (CatL) in puromycin aminonucleoside-induced injury of mouse glomerular podocytes.
METHODSMouse podocyte cells (MPCs) in vitro cultured were divided into the normal control group, the model group, the dexamethasone (DEX) group, 10% WHLR containing serum group, 20% WHLR containing serum group, the vehicle serum control group. MPCs in the normal control group were cultured at 37 degrees C culture solution for 24 h. 45 mg/L puromycin was acted on MPCs in the model group for 24 h. On the basis of puromycin intervention, 1 limol/L DEX was co-incubated in MPCs of the DEX group for 24 h; 10% or 20% WHLR containing serum was co-incubated in MPCs of the 10% WHLR containing serum group and 20% WHLR containing serum group for 24 h. The vehicle serum control group was also set up by incubating with WHLR containing serum alone for 24 h. The expression of CatL and its substrate Synaptopodin in podocytes were detected by cell immunofluorescence staining. FITC-conjugated phalloidin was used to stain F-actin. A cortical F-actin score index (CFS index) was designed to quantify the degree of cytoskeletal reorganization in cultured podocytes.
RESULTSCompared with the normal control group, the expression of synaptopodin significantly decreased and the expression of CatL significantly-increased in the model group. F-actin arranged in disorder, gradually forming pericellular F-actin ring. CFS index was obviously elevated (P < 0.01). Compared with the model group, the epression of synaptopodin increased, the expression of CatL decreased, and CFS index also decreased in the DEX group, 10% WHLR containing serum group, and 20% WHLR containing serum group (P < 0.05, P < 0.01). Compared with the DEX group, the expression of synaptopodin decreased in 10% WHLR containing serum group, CFS index also decreased in 20% WHLR containing serum group (P < 0.05).
CONCLUSIONSWHLR could up-regulate the expression of synaptopodin, down-regulate the expression of CatL, and alleviate cytoskeletal reorganization of F-actin. It was helpful to stabilize the cytoskeleton of F-actin and improve the merging of podocytes.
Actins ; metabolism ; Animals ; Cathepsin L ; metabolism ; Cells, Cultured ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Kidney Glomerulus ; cytology ; Mice ; Microfilament Proteins ; metabolism ; Podocytes ; drug effects ; pathology ; Puromycin Aminonucleoside ; adverse effects ; Up-Regulation
4.Angiotensin II Modulates p130Cas of Podocytes by the Suppression of AMP-Activated Protein Kinase.
Tae Sun HA ; Hye Young PARK ; Su Bin SEONG ; Hee Yul AHN
Journal of Korean Medical Science 2016;31(4):535-541
Angiotensin II (Ang II) induces the pathological process of vascular structures, including renal glomeruli by hemodynamic and nonhemodynamic direct effects. In kidneys, Ang II plays an important role in the development of proteinuria by the modification of podocyte molecules. We have previously found that Ang II suppressed podocyte AMP-activated protein kinase (AMPK) via Ang II type 1 receptor and MAPK signaling pathway. In the present study, we investigated the roles of AMPK on the changes of p130Cas of podocyte by Ang II. We cultured mouse podocytes and treated them with various concentrations of Ang II and AMPK-modulating agents and analyzed the changes of p130Cas by confocal imaging and western blotting. In immunofluorescence study, Ang II decreased the intensity of p130Cas and changed its localization from peripheral cytoplasm into peri-nuclear areas in a concentrated pattern in podocytes. Ang II also reduced the amount of p130Cas in time and dose-sensitive manners. AMPK activators, metformin and AICAR, restored the suppressed and mal-localized p130Cas significantly, whereas, compound C, an AMPK inhibitor, further aggravated the changes of p130Cas. Losartan, an Ang II type 1 receptor antagonist, recovered the abnormal changes of p130Cas suppressed by Ang II. These results suggest that Ang II induces the relocalization and suppression of podocyte p130Cas by the suppression of AMPK via Ang II type 1 receptor, which would contribute to Ang II-induced podocyte injury.
AMP-Activated Protein Kinases/antagonists & inhibitors/chemistry/*metabolism
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Aminoimidazole Carboxamide/analogs & derivatives/pharmacology
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Angiotensin II/*pharmacology
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Angiotensin II Type 1 Receptor Blockers/pharmacology
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Animals
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Blotting, Western
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Cell Line
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Cell Nucleus/metabolism
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Crk-Associated Substrate Protein/*metabolism
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Cytoplasm/metabolism
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Focal Adhesion Kinase 1/metabolism
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Losartan/pharmacology
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Metformin/pharmacology
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Mice
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Microscopy, Confocal
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Podocytes/cytology/drug effects/metabolism
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Protein Kinase Inhibitors/*pharmacology
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Ribonucleotides/pharmacology
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Signal Transduction/*drug effects