ABSTRACT
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.
Subject(s)
Animals , Mice , AMP-Activated Protein Kinases/antagonists & inhibitors , Aminoimidazole Carboxamide/analogs & derivatives , Angiotensin II/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Crk-Associated Substrate Protein/metabolism , Cytoplasm/metabolism , Focal Adhesion Kinase 1/metabolism , Losartan/pharmacology , Metformin/pharmacology , Microscopy, Confocal , Podocytes/cytology , Protein Kinase Inhibitors/pharmacology , Ribonucleotides/pharmacology , Signal Transduction/drug effectsABSTRACT
PURPOSE: Puromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro. METHODS: We observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction. RESULTS: In the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants. CONCLUSION: These findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.
Subject(s)
Animals , Mice , Rats , Actin Cytoskeleton , Actins , Antioxidants , Ascorbic Acid , Blotting, Western , Cadherins , Catechin , Crk-Associated Substrate Protein , Cytoplasm , Cytoskeleton , Diaphragm , Epithelial Cells , Fluorescent Antibody Technique , Foot , Glomerular Basement Membrane , Oxidative Stress , Podocytes , Probucol , Proteins , Puromycin , Puromycin Aminonucleoside , RNA, MessengerABSTRACT
BACKGROUND: BCAR1/p130Cas protein is the human homologue of rat p130Cas protein, and it is a docking protein involved in the intracellular signaling pathways. This protein also causes the proliferating human breast cancer cells to be resistant to antiestrogen drugs. The fragile histidine triad (FHIT) protein is presumed to have a tumor suppressor function in a number of human tumors. The aim of this study was to investigate expressions of p130Cas and FHIT in breast carcinomas and to evaluate their relationship with the clinicopathological prognostic factors. METHODS: A total of 93 cases of invasive breast carcinomas was retrospectively reviewed. The expressions of p130Cas and FHIT were examined by immunohistochemical methods. RESULTS: p130Cas expression was observed in all breast carcinomas: p130Cas immunoreactivity was strongly positive in 39 cases (41.9%), moderately positive in 49 cases (52.7%) and weakly positive in 5 cases (5.4%) of 93 cases. It was statistically correlated with the p53 (p=0.035) and c-erbB-2 (p=0.024) expressions. The FHIT protein expression was markedly reduced or completely negative in 59 cases (63.4%), but it was not correlated with the clinicopathological prognostic factors. There was no significant correlation between p130Cas and FHIT expressions. CONCLUSIONS: This study seems to provide meager information on whether these proteins may be useful prognostic factors, and so this topic needs further study.