AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway

Li XC, Hopfer U, Zhuo JL. AT(1) receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway. Am J Physiol Renal Physiol 297: F1342-F1352, 2009. First published September 2, 2009; doi: 10.1152/ajprenal.90734.2008.-Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT(1) (AT(1a)) 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 AT(1)-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT(1) receptors, 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 AT(1)-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT(1) siRNAs inhibited AT(1) expression 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 similar to 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 AT(1)-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 similar to 52% decreases in AT(1)-mediated FITC-ANG II uptake and similar to 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 AT(1) expression or clathrin-dependent transferrin uptake. Both losartan and AT(1a) receptor 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 AT(1) (AT(1a))mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.