AT1 and AT2 receptors mediate opposing mitochondrial respiratory and glycolytic stress responses to mitochondrial angiotensin II in proximal tubule cells
Jia L. Zhuo & Xiao C. Li. Laboratory of Receptor & Signal Transduction, Department of Pharmacology and Toxicology, Division of Nephrology, and Center of Excellence for Cardiovascular and Renal Research, The University of Mississippi Medical Center, Jackson, Mississippi, USA (jzhuo@umc.edu)
Angiotensin II (ANG II) plays an important role in mediating mitochondrial dysfunction in cardiovascular, hypertensive and renal diseases, but whether extracellular or intracellular ANG II mediates mitochondrial dysfunction via cell membrane or mitochondrial ANG II receptors remains poorly understood. In this proof of concept study, we overexpressed an intracellular ANG II fusion protein, mito-ANG II, with or without a full length ANG II type 2 receptor, mito-AT2R, in the mitochondria of mouse proximal tubule cells (mPCT). The mitochondrial respiratory and glycolytic stress responses were measured using Seahorse XF Cell Mito and XF Glycolysis Stress Test Kits, respectively. Live cell fluorescent imaging confirmed colocalization of mito-ANG II, mito-AT2R, and a mitochondrial marker MitoTracker®. Overexpression of mito-ANG II for 48 h markedly increased oxygen consumption rate (OCR) by ~29% (Control: 239.4 ± 9.2 vs. mito-ANG II: 310.4 ± 12.6 pmol/min; p<0.01, n=5) and extracellular acidification rate (ECAR) by 33% (Control: 6.3 ± 0.3 vs. mito-ECFP/ANG II: 8.1 ± 0.5 mpH/min; p<0.01, n=5). The effects of mito-ANG II on OCR and ECAR were associated with increases in phosphorylated MAP kinases ERK1/2, Na+/K+-ATPase, and mitochondrial redox carries, Complex I (NADH coenzyme Q reductase), Complex II (succinate dehydrogenase), Complex III (cytochrome bc1 complex) and Complex IV proteins (cytochrome c oxidase) (p<0.01, n=6). The mito-ANG II-induced OCR and ECAR responses were blocked by the AT1 blocker losartan (10 µM, p<0.01, n=5), but not the AT2 receptor blocker PD123319 (10 µM, n.s., n=5). However, concurrent overexpression of mito-AT2R with mito-ANG II in mPCT cells significantly attenuated the effects of mito-ANG II on OCR (p<0.01, n=6) and ECAR (p<0.01, n=6). PD123319 blocked the effects of mito-AT2R overexpression on OCR (p<0.05, n=5) and ECAR (p<0.01, n=5). Taken together, our results strongly support the proof of concept that activation of mitochondrial AT1 receptors by ANG II stimulates, whereas activation of mitochondrial AT2 receptors by mitochondrial ANG II inhibits, mitochondrial respiratory and glycolytic responses in mouse proximal tubule cells.
(Supported by 2R01DK067299, 2R01DK102429 & R56HL130988)
