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RECEPTORS AND SIGNAL TRANSDUCTION

Selective knockdown of AT₁ receptors by RNA interference inhibits Val⁵-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells

¹Laboratory of Receptor and Signal Transduction, Division of Hypertension and Vascular Research, Henry Ford Hospital and ²Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan

Submitted 29 August 2006 ; accepted in final form 31 March 2007

Receptor-mediated endocytosis of extracellular ANG II has been suggested to play an important role in the regulation of proximal tubule cell (PTC) function. Using immortalized rabbit PTCs as an in vitro cell culture model, we tested the hypothesis that extracellular ANG II is taken up by PTCs through angiotensin type 1 receptor (AT₁; or AT_1a) receptor-mediated endocytosis and that inhibition of ANG II endocytosis using a selective AT₁ receptor small-interfering RNA (siRNA; AT₁R siRNA) or endocytotic inhibitors exerts a physiological effect on total and apical sodium and hydrogen exchanger isoform 3 (NHE-3) protein abundance. Western blots and live cell imaging with FITC-labeled ANG II confirmed that transfection of PTCs with a human specific AT₁R siRNA for 48 h selectively knocked down AT₁ receptor protein by 76 ± 5% (P < 0.01), whereas transfection with a scrambled siRNA had little effect. In nontransfected PTCs, exposure to extracellular ANG II (1 nM) for 60 min at 37°C increased intracellular ANG II accumulation by 67% (control: 566 ± 55 vs. ANG II: 943 ± 160 pg/mg protein, P < 0.05) and induced mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (163 ± 15% of control, P < 0.01). AT₁R siRNA reduced ANG II endocytosis to a level similar to losartan, which blocks cell surface AT₁ receptors (557 ± 37 pg/mg protein, P < 0.05 vs. ANG II), or to colchicine, which disrupts cytoskeleton microtubules (613 ± 12 pg/mg protein, P < 0.05 vs. ANG II). AT₁R siRNA, losartan, and colchicine all attenuated ANG II-induced ERK1/2 activation and total cell lysate and apical membrane NHE-3 abundance. The scrambled siRNA had no effect on ANG II endocytosis, ERK1/2 activation, or NHE-3 expression. These results suggest that AT₁ receptor-mediated endocytosis of extracellular ANG II may regulate proximal tubule sodium transport by increasing total and apical NHE-3 proteins.

extracellular signal-regulated kinase 1/2; kidney; sodium transport; receptor internalization; ribonucleic acid interference

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