Abstract 478: Is Ang II-mediated mTOR/S6K1 Activation in Proximal Tubule Cells mTORC2-dependent?

Abstract

Introduction: Activation of the mTOR nutrient sensing pathway in early diabetes has been implicated in glomerulomegaly, hyperfiltration and hypertrophy of the nephrons. Recent studies show that excess/deletion of mTOR/raptor (mTORC1) in the podocytes results in diabetic changes. Interestingly, mTOR exists in another complex called mTORC2 (mTOR/rictor), which has been shown to phosphorylate Akt at S473 and is proposed to regulate cell motility and actin cytoskeleton. Recently, growth factors such as angiotensin II (Ang II) have been identified as activators of mTORC1 in cardiomyocytes, intestinal epithelial cells and embryonic cell. However, the precise mechanism of mTORC1 activation in the proximal tubule cells (PTC) is largely unknown. We hypothesized that Ang II activation of mTORC1 is dependent on mTORC2 activation. Methods: Mice were subjected to Ang II (200ng/kg/min) via osmotic minipump and whole kidney lysates analyzed for mTORC1 activation. In parallel, acute Ang II (10 -7 M, 10min) treatment of opossum PTCs stably expressing rat AT1 B R was carried out on 24 hour starved cultures preceded by 1 hour pretreatment with various inhibitors. Results: Ang II activated mTORC1/S6 kinase in kidney lysates (1.5-fold) and cultured PTCs (3-5 fold) as evidenced by increased phosphorylation of T389-S6K1, S240/244-RPS6 and T36/45-4EBP1. Ang II also activated mTORC2 (2-fold) as evidenced by p-S473-Akt. Upstream, Ang II caused phosphorylation of EGF receptor and inhibition of EGFR activation resulted in 30% inhibition of Akt activation and partial inhibition of mTOR/S6 kinase. Inhibition of PKC also resulted in partial inhibition of mTOR/S6 activation but the magnitude (50%) was higher than inhibition of EGFR signaling. Rapamycin (10nM) inhibited mTORC1 activation and not mTORC2 but PP242 (ATP site inhibitor, 8nM) completely abolished both mTORC1 and mTORC2 activation. Conclusion: Ang II activation of mTOR/S6K follows a distinct pattern in PTC when compared to other cell types. Ang II induced activation of mTOR/S6 kinase is mediated by both PKC and EGFR activation. mTORC2 appears to play a central role in regulating Ang II induced activation of mTOR/S6 kinase.