Nuria preserved glomerular Whose structure and further support the activation of Ras in diabetes. PHA-680632 Previous studies from our laboratory have shown fa A constant high glucose Ang II production in mesangial cells mainly through the synthesis of increasingly Agt, the precursor Shore of Ang II activated. In addition, lead exposure of mesangial cells high glucose increased Ht of angiotensin II in the cell lysates, the significant h Ago as extracellular Were re Ang II levels found in the cell media. In addition, our studies have shown that previous inhibition of extracellular Ren Ang II formation to a partial blockage of the Erh Increase of high glucose in TGF b1 and matrix induces, whereas after suppression of both the intracellular Re formation and extracellular Ren Ang II by knockdown produced a gr ere inhibition of TGF-b1 and matrix AGT.
These results led to the hypothesis that intracellular Ang II may contribute re the overall increase in TGF b1 and matrix proteins in mesangial high glucose condition. Therefore, this study was con Ue to determine whether intracellular Re Ang II k can Independently Ngig influence each other TGF b1 and matrix in mesangial cells, without Bergenin the participation of the signal path of the extracellular Ren Ang II Cultured human mesangial cells were treated with Ang II-transfected to the intracellular re-Ang II levels increased hen, w during candesartan was used to activate the extracellular Ren to block Ang II signaling via AT1-receptor on the cell membrane.
The results of this study suggest that Ang II may be intracellular Ren TGF b1 and increased mesangial matrix hen And also activates the transcription factor STAT3 independent Ngig of the extracellular Ren matrix Ang II signaling pathway. 2.Methods 2.1. Chemicals. Angiotensin II was obtained from Sigma Chemicals and angiotensin-II-fluorescein-conjugated purchased from Invitrogen. AG-490 and Jak inhibitor I were from Calbiochem. SDS, acrylamide / BIS nitrocellulose membrane, Tween 20, ammonium persulfate, TEMED and protein assay reagents were purchased from Bio Rad Laboratories, and other reagents from Sigma Chemicals. Anti-total Stat3, actin and goat anti-rabbit horseradish peroxidase-conjugated IgG were from Cell Signaling Technology and Jak2 Antique Get body from Chemicon. The molecular weight marker protein was obtained from Amersham, and the chemiluminescence kit from Pierce.
Candesartan was obtained from Astra Zeneca Pharmaceuticals. 2.2. Culture of human mesangial cells. Prim Normal human mesangial cells were re receive from ScienCell channel 1 and maintained in growth media of mesangial cells with 5% f Fetal bovine serum serumand 1 g / ml gentamicin at 37 C in 5% CO 2 and 95% air. The cells were subcultured at 70 80% confluence, and experiments were performed with cells between passages 2 and 5. 2.3. Transfection of cells with Ang II To the R The intracellular Ren Ang II to study specifically, increases the intracellular ht Higher concentrations of Ang II with a protein transfection reagent. 10 dilution with serum-free and complements a MSGM: In summary, Ang II with proteomic juice was following the instructions of the manufacturer and incubated mixed for 20 minutes at room temperature, then first Mesangial cells were then incubated with this medium for 20 minutes to 24 hours, depending on the experimental protocol. Inhibit the binding of all free Ang II in the mixture in the cell proteo juice