AT1receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway

Abstract

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1(AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1siRNAs inhibited AT1expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected ( P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by ∼90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin ( P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in ∼52% decreases in AT1-mediated FITC-ANG II uptake and ∼66% decreases in ANG II-induced NHE-3 expression ( P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation ( P < 0.01), but not with altered AT1expression or clathrin-dependent transferrin uptake. Both losartan and AT1areceptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1(AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.