Abstract 009: Mitochondria-targeting Overexpression of AT 2 Receptors Inhibits Intracellular Angiotensin II-induced Respiratory and Glycolytic Stress Responses in Mouse Proximal Tubule Cells

Abstract

Angiotensin II (ANG II) plays an important role in mitochondrial dysfunction associated with cardiovascular, hypertensive and kidney diseases, but it is not known whether extracellular or intracellular ANG II mediates this effect via activation of cell surface or mitochondrial receptors. As a proof of concept study, we overexpressed an intracellular cyan fluorescent ANG II fusion protein, mito-ANG II, with or without a GFP-tagged full length AT 2 receptor, mito-AT 2 R, selectively in the mitochondria of mouse proximal tubule (mPCT) cells. 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 the expression and colocalization of mito-ANG II and mito-AT 2 R with a mitochondrial marker MitoTracker®. Overexpression of mito-ANG II for 48 h significantly increased mitochondrial oxygen consumption rate (OCR) by 30% (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-ANG II: 8.1 ± 0.5 mpH/min; p <0.01, n=5). The effects of mito-ANG II on OCR and ECAR responses were associated with significant increases in phosphorylated MAP kinase ERK1/2, Na + /K + -ATPase, and mitochondrial redox carries, Complex I (NADH coenzyme Q reductase), Complex II (succinate dehydrogenase), Complex III (cytochrome bc 1 complex) and Complex IV (cytochrome c oxidase) ( p <0.01, n=6). The mito-ANG II-induced OCR and ECAR responses were blocked by the AT 1 blocker losartan (10 μM, p <0.01, n=5), but not by the AT 2 receptor blocker PD123319 (10 μM, n.s. , n=5). However, concurrent overexpression of mito-AT 2 R with mito-ANG II in the mitochondria of mPCT cells significantly attenuated the effects of mito-ANG II on OCR and ECAR responses ( p <0.01, n=6), while the effects of mito-AT 2 R overexpression were completely blocked by PD123319 ( p <0.01, n=5). Taken together, our results provide strong evidence that activation of mitochondrial AT 1 receptors by intracellular ANG II stimulates, whereas activation of mitochondrial AT 2 receptors by intracellular ANG II inhibits, mitochondrial respiratory and glycolytic responses in mouse proximal tubule cells.