Role of Angiotensin II internalization in the activation of Proximal Tubule Angiotensinogen

Angiotensin (Ang) II increases the expression of angiotensinogen (AGT) mRNA and protein by proximal tubules (PT) in the kidney; however the molecular mechanisms are unclear. Because Ang II internalization regulates several actions of this hormone in PT cells, studies were performed to determine if Ang II internalization is required for activation of AGT expression in these cells. Immortalized rat PT cells (IRPTCs) were exposed to Ang II (1 nM, 1 hour), with or without an AT1R blocker (candesartan, 10 μM), or an endocytosis inhibitor (PAO, 0.1 μM) before mRNA collection. AGT mRNA expression was determined by qRT‐PCR. Ang II internalization was determined by exposing cells to fluorescent‐Ang II (Alexa488‐Ang II, 100 nM, 15–240 min) and detecting intracellular fluorescent signal with an epifluorescence microscope. Ang II increased AGT expression by 20% (1.21 ± 0.19 vs. 1.00 in controls, p<0.05). This effect was both AT1R‐dependent and endocytosis‐dependent as it was prevented by candesartan (0.89 ± 0.24, NS) and by PAO respectively (0.93 ± 0.04, NS). Microscopy studies demonstrated that Ang II is internalized in a time‐dependent manner, with a signal shift from the cell membrane to the perinuclear area after 1 hour. In conclusion, Ang II internalization by PT cells is required for activation of the AGT gene, and this effect is concomitant with a signal shift that suggests Ang II translocation to perinuclear compartments. Work supported by NIH grants P20RR017659, HL26371 (LGN), (R01DK072408) (HK), and the COSEHC Warren Trust Fellowship award (RAGV).