Abstract 487: AT1a Receptor-Mediated Uptake of Extracellular Angiotensin II and NHE3 Expression in Mouse Proximal Tubule Cells partly Involve the Multi-ligand Endocytic Receptor Megalin

Abstract

Megalin, the multi-ligand endocytic receptor abundantly expressed in apical (AP) membranes of proximal tubules, plays a crucial role in mediating the uptake of low molecular weight (LMW) proteins in the kidney. Deletion of megalin leads to the development of LMW proteinuria in mice. In the present study, we tested the hypothesis that megalin in AP membranes mediates the uptake (or endocytosis) of angiotensin II (ANG II) in mouse proximal tubule cells (mPCT) by interacting with AT 1a receptors. Semi-confluent, polarized monolayers of mPCT cells of wild-type (WT) and AT 1a -deficient (AT 1a -KO) mice grown on transwell permeable supports or 6-well plates were treated from the AP surface with vehicle, losartan (10 μM), PD123319 (10 μM), a selective megalin-siRNA or a scrambled siRNA for 48 h. The time-dependent uptake of fluorescein (FITC)-labeled ANG II (10 nM, 37 o C) was then determined by fluorescence imaging. As expected, AT 1a receptor and megalin proteins were abundantly expressed in AP membranes of WT mPCT cells, whereas AT 1a receptors were absent in AT 1a -KO mPCT cells ( p <0.01 vs. WT cells). In WT mPCT cells, the uptake of FITC-ANG II was peaked at 30 min and visualized in the nuclei by 1 h. These responses were blocked by losartan ( p <0.01), whereas PD123319 had no effect. Furthermore, AT 1a receptor-mediated FITC-ANG II uptake was largely blocked in AT 1a -KO mPCT cells ( p <0.01 vs. WT cells). In both WT and AT 1a -KO mPCT cells, the specific megalin-siRNA knocked down 86.3±5.2% of megalin protein expression ( p <0.01), but it inhibited only 32.8±3.1% of FlTC-ANG II uptake in WT mPCT cells ( p <0.01 vs.losartan). Megalin-siRNA had no effect on FITC-ANG II uptake in AT 1a -KO mPCT cells. The AT 1a receptor-mediated uptake of FITC-ANG II was associated with 3-fold increases in phosphorylated MAP kinases ERK1/2 proteins (control: 0.36±0.06 vs. ANG II: 1.07±0.10, p <0.001) and 1-fold increase in p-NHE3 proteins (control: 0.18±0.03 vs. ANG II: 0.37±0.04, p <0.01) in WT mPCT cells, which was blocked by losartan (0.17±0.07, p <0.01 vs. ANG II) and megalin-siRNA (0.19±0.02 p <0.01 vs. ANG II), respectively. These results suggest that AT 1a receptor-mediated uptake of extracellular Ang II and NHE3 expression in mPCT cells may partly involve the multi-ligand endocytic receptor megalin.