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

• Published in: American Journal of Physiology - Renal Physiology 2009-11, Vol.297 (5), p.F1342-F1352
• Main Authors: Li, Xiao C, Hopfer, Ulrich, Zhuo, Jia L
• Format: Article
• Language: English
• Subjects: Actins - biosynthesis; Actins - genetics; Angiotensin II - metabolism; Angiotensin II - pharmacology; Animals; Blotting, Western; Cells, Cultured; Clathrin - biosynthesis; Cytoskeletal Proteins - biosynthesis; Cytoskeletal Proteins - genetics; Endocytosis - physiology; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Indoles; Kidney Tubules, Proximal - cytology; Kidney Tubules, Proximal - drug effects; Kidney Tubules, Proximal - metabolism; Mice; Microscopy, Fluorescence; Microtubules - drug effects; Microtubules - metabolism; Rabbits; Receptor, Angiotensin, Type 1 - biosynthesis; Receptor, Angiotensin, Type 1 - genetics; Receptor, Angiotensin, Type 1 - physiology; RNA, Small Interfering - pharmacology; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers - biosynthesis; Sodium-Hydrogen Exchangers - genetics
• ISSN: 0363-6127; 1522-1466
• DOI: 10.1152/ajprenal.90734.2008

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 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 approximately 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 approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 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 AT1a 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 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.