Abstract P611: Immortalization of Sheep Proximal Tubule Cells Retain the Ability to Internalize Angiotensinogen that Traffics to the Mitochondria and Nucleus

Abstract

Although there is compelling evidence for an intracellular renin-angiotensin system (RAS) that includes localization of AT1, AT2 and AT7/Mas receptors (R) on the nucleus and mitochondria of various cell types, the mechanism for the intracellular expression of angiotensins remains equivocal as the precursor protein angiotensinogen (Aogen) enters the secretory pathway upon synthesis. Proximal tubules (PTs) of the kidney present a unique cell system since the PTs internalize Aogen and transgenic mice lacking either the PT protein transporter megalin or liver Aogen exhibit reduced renal content of both Aogen and Ang II. We reported that isolated sheep PTs readily internalize Aogen, and subcellular fractionation revealed that Aogen was evident in the nuclear and mitochondrial fractions. The present study sought to establish a permanent cell line derived from the sheep PT to facilitate the characterization of Aogen internalization and processing. Sheep PT cells were isolated by protease digestion and Percoll density gradient separation, maintained in culture to promote epithelial cell growth and immortalized by SV-40 transfection. A clone (SPT-1) was obtained that expressed the SGLT-2 protein, a selective PT marker. SPT-1 cells were incubated with recombinant 125 I-Aogen at 37°C in DMEM/F12 media. A time course [0.5 to 6 hrs] revealed linear uptake of Aogen [r = 0.995] that did not saturate by 6 hrs. Pre-treatment of the SPT-1 cells with renin/ACE/neprilysin/chymase inhibitors [INHIB] or AT1R/AT2R/AT7/MasR antagonists [ANTAG] failed to attenuate Aogen internalization [Control: 209 ± 22; INHIB: 200 ± 21; ANTAG: 217 ± 15 fmol/hr/mg, n=3] while Ang II or Ang-(1-7) [10 μM, each] also did not inhibit, but tended to increase Aogen uptake [238 ± 24 and 244 ± 15 fmol/hr/mg, respectively, n=3]. Subcellular fractionation studies revealed that 12.0 ± 0.2% [n=3] of the total internalized Aogen was localized to the mitochondrial fraction with a higher content in the nucleus following an 18 hr uptake. We conclude that the established SPT-1 cell line which retains the capacity to internalize Aogen and expresses a similar pattern of protein trafficking to isolated PTs, may constitute a relevant model to elucidate the pathway for intracellular expression of angiotensins.