The present study tested the hypothesis that the multiligand endocytic receptor megalin is partially involved in the uptake of ANG II and downstream signaling responses in mouse proximal tubule cells (mPCT) by interacting with AT1a receptors. at 30 min in the cytoplasm and in the nuclei 1 h after exposure. Losartan alone completely blocked the uptake of FITC-ANG II, whereas megalin siRNA inhibited only 30% of the response (< 0.01). The remaining FITC-ANG II uptake in the presence of megalin siRNA was completely abolished by losartan. ANG II induced threefold increases in phosphorylated MAP kinases ERK1/2 and a onefold increase in phosphorylated sodium and hydrogen exchanger 3 (NHE3) protein, which were also blocked by losartan and megalin-siRNA. By contrast, losartan and megalin siRNA had no effects on these signaling proteins in AT1a-KO mPCT cells. We conclude that the uptake of ANG II and downstream MAP kinases ERK1/2 and NHE3 signaling responses in mPCT cells are mediated primarily by AT1a receptors. However, megalin may also play a partial role in these responses to ANG II. were subcultured to 80% confluence in six-well plates or glass coverslips, as appropriate, in the complete DMEM/F-12 growth medium at 37C, supplied with 95% O2-5% CO2, 50 nM hydrocortisone, 5% heat-inactivated FBS, 100 U/ml penicillin, and 100 g/ml streptomycin, as we described previously (27, 30, 33, 47). Untreated wild-type and AT1a-KO mPCT cells were used as controls in all experiments. AT1a receptor expression in mPCTs. The expression of AT1 (AT1a) receptors in wild-type mPCT cells or the lack of AT1a receptor expression in AT1a-KO mPCT cells was recently characterized using [125I]-ANG II receptor binding assays, RT-PCR, and Western blotting, respectively (25, 31, 32, 47). Immunofluorescent imaging and Western blot analysis of megalin protein expression in mPCT cells. mPCT cells were split cultured on glass coverslips to 80% confluence. After the medium was removed and mPCTs were washed with PBS twice, the cells were first incubated with a specific megalin primary antibody (sc-16476, 1:100) for 3 h, followed by incubation with a secondary donkey anti-goat FITC-labeled antibody (sc-2024, 1:100) for 3 h, respectively, for immunofluorescent imaging of megalin proteins, as we described (30). For unfavorable control of megalin immunofluorescence, mPCT cells grown on glass coverslips were incubated only with the Mouse monoclonal to GST donkey anti-goat FITC-IgG without a Telithromycin (Ketek) manufacture first incubation with a primary megalin antibody. A blocking peptide was also previously used to verify the specificity of the first megalin antibody (30). Megalin immunofluorescence was visualized using a Nikon-Eclipse TE2000-U inverted fluorescence microscope and a FITC-specific filter (26, 27, 30). Western blot analysis was also performed to quantitate megalin protein expression in WT and AT1a-KO mPCTs. Knockdown of megalin expression in mPCT cells. To determine the role of megalin in mediating ANG II or AT1a-dependent uptake of ANG II in mPCT cells, wild-type or AT1a-KO mPCTs were transfected with a selective 19- to 25-nucleotide megalin siRNA (sc-40103, 4 g/well), or a scrambled siRNA (sc-37007, 4 g/well) for 48 h using Lipofectamine 2000 (27, 30). The specificity and the effectiveness of Telithromycin (Ketek) manufacture megalin siRNA in knocking down megalin expression were decided by measuring megalin mRNA expression in mPCT cells by RT-PCR and live cell fluorescent imaging of megalin expression. The forward and reverse primers for PCR of megalin mRNA expression were provided by Santa Cruz Biotechnology (sc-40103-PR), and the annealing and extending temperatures were 55-60C and 68-70C, respectively (24). The effect of specific megalin siRNA on megalin protein knockdown was further decided using a specific megalin primary antibody (sc-16476, 1:100). Live cell fluorescent imaging of megalin- and AT1a receptor-mediated FITC-labeled ANG II in mPCT cells. Wild-type and AT1a-KO mPCT cells were produced to 60% confluence on glass coverslips. Under these conditions, mPCT cells did not have the entire lateral and basal membranes uncovered to ligands or drugs. Only brush border or apical membranes were uncovered to ligands or drugs. Untreated, the AT1 receptor antagonist losartan-, AT2 receptor antagonist PD123319-, megalin siRNA-, or scrambled Telithromycin (Ketek) manufacture siRNA-treated wild-type and AT1a-KO mPCT cells were incubated with FITC-ANG II (1 nM, Molecular Probes) at 37C without exposure to light for 30 min to.