Selective knockdown of AT1 receptors by RNA interference inhibits Val5-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells.
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 (AT(1); or AT(1a)) receptor-mediated endocytosis and that inhibition of ANG II endocytosis using a selective AT(1) receptor small-interfering RNA (siRNA; AT(1)R 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 AT(1)R siRNA for 48 h selectively knocked down AT(1) 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 degrees 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). AT(1)R siRNA reduced ANG II endocytosis to a level similar to losartan, which blocks cell surface AT(1) 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). AT(1)R 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 AT(1) receptor-mediated endocytosis of extracellular ANG II may regulate proximal tubule sodium transport by increasing total and apical NHE-3 proteins.
Li
Xiao C
XC
Div. of Hypertension and Vascular Research, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202, USA.
Zhuo
Jia L
JL
D000804:Angiotensin II / Q000378:metabolism*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003078:Colchicine / Q000494:pharmacology; D003080:Cold Temperature; D015536:Down-Regulation; D004705:Endocytosis / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019808:Losartan / Q000494:pharmacology; D008870:Microtubules / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D044140:Receptor, Angiotensin, Type 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D050257:Tubulin Modulators / Q000494:pharmacology
D047228:Angiotensin II Type 1 Receptor Blockers; D034741:RNA, Small Interfering; D044140:Receptor, Angiotensin, Type 1; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D050257:Tubulin Modulators; D000804:Angiotensin II; D012964:Sodium; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019808:Losartan; D003078:Colchicine