Objective: Sirtuin 3 (Sirt3) in the mitochondrial matrix plays an important protective role against angiotensin II-induced hypertension and kidney injury. However, the mechanisms underlying its anti-oxidative, anti-inflammation, and anti-hypertensive effects remain incompletely understood. The present study tested the hypothesis that overexpression of NAD-dependent deacetylase sirtuin 3 selectively in the mitochondria attenuates intracellular angiotensin II (Ang II)-induced mitochondrial dysfunction in proximal tubule (mPCT) cells. Design and method: To test the hypothesis, an adenovirus-mediated, mitochondria-targeting construct, Ad-Sglt2-mito-Sirt3, was custom-designed, amplified, and purified for overexpression of Sirt3 selectively in the mitochondria of mPCT cells (Vector BioLabs, Malvern, PA). mPCT cells were cultured in 6-well plates for 48 h and treated with: a) control construct (C, n=10); b) overexpression of Ad-Sglt2-mito-Sirt3 alone (Sirt3, n=10); c) overexpression of an mitochondria-targeting intracellular angiotensin fusion protein, Ad-Sglt2-mito-ECFP/Ang II alone (mito-Ang II, n=10); or d) concurrent overexpression of Ad-Sglt2-mito-ECFP/Ang II and Ad-Sglt2-mito-Sirt3 (mito-Ang II+Sirt3, n=10). The expression of mitochondrial electron transport proteins and Sirt3 in mPCT cells was determined by Western blot analysis, whereas mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using Seahorse XFe 24 Extracellular Flux Analyzer, respectively. Results: Compared with control mPCT cells, Sirt3 proteins were markedly increased by 2-fold in mPCT cells overexpressing Ad-Sglt2-mito-Sirt3 (P< 0.01). Overexpression of Ad-Sglt2-mito-ECFP/Ang II significantly increased the expression of major mitochondrial electron transport proteins, CII-CV, in mPCT cells (P< 0.01), which was markedly attenuated by the AT1 receptor blocker losartan (10 microM) or concurrent overexpression of Ad-Sglt2-mito-Sirt3 (P< 0.01). Ad-Sglt2-mito-ECFP/Ang II-induced expression of mitochondrial electron transport proteins was associated with marked increases in OCR and ECAR responses in mPCT cells, and these responses were completely blocked by losartan or overexpression of Ad-Sglt2-mito-Sirt3 (P< 0.01). Although AT2 receptor blocker PD123319 had no effect on Ad-Sglt2-mito-ECFP/Ang II-induced OCR and ECAR responses, overexpression of Ad-Sglt2-mito-AT2 receptors, however, significantly attenuated Ad-Sglt2-mito-ECFP/Ang II-induced OCR and ECAR responses in mPCT cells (P< 0.01). Conclusions: Taken together, these results support the hypothesis that NAD-dependent deacetylase sirtuin 3 in the mitochondria of mPCT cells plays a protective role against mitochondrial Ang II-induced mitochondrial dysfunction in mPCT cells.
Zhuo et al. (Fri,) studied this question.