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This Article Full Text Full Text (PDF) All Versions of this Article: 297/5/F1342    most recent 90734.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Li, X. C. Articles by Zhuo, J. L. PubMed PubMed Citation Articles by Li, X. C. Articles by Zhuo, J. L.

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

¹Laboratory of Receptor and Signal Transduction, Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit; ; ²Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio; and ; ³Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan

Submitted December 8, 2008 ; accepted in final form August 26, 2009

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT₁ (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 AT₁-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT₁ 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 AT₁-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT₁ siRNAs inhibited AT₁ 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 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 AT₁ 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 AT₁ (AT_1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.

kidney; clathrin-coated pits; cytoskeleton; G protein-coupled receptor; MAP kinases; small interfering RNA