Angiotensinogen import in isolated proximal tubules: evidence for mitochondrial trafficking and uptake

Abstract

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenousI-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; = 4). Subsequent studies determined whether mitochondria directly internalized I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalizedI-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min·mg protein; = 3). Lastly, unlabeled angiotensinogen (100 nM) competed forI-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; < 0.05, = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

Angiotensin dependent and angiotensin independent protective effects of renin-b in H9c2 cells after anoxia

“…However, whereas internalization has been demonstrated 30 , a mechanism of how ANGII may leave the vesicles is not known so far. Also, angiotensinogen appears to be internalized 31 . On the other hand, Singh et al demonstrated that ANG can be generated intracellularly in the absence of extracellular components of the RAS in neonatal cardiomyocytes, and that the system can be stimulated by high glucose 32 .…”

The intracellular renin-angiotensin system: Friend or foe. Some light from the dopaminergic neurons

“…The possible presence of a mitochondrial RAS was suggested after observing different RAS components such as renin, ACE, and Ang II in cells from adrenal gland, kidney or cerebellar cortex, particularly using electron microscopy or uptake of labelled Ang II studies ( Erdmann et al, 1996 ; Peters et al, 1996 ; van Kats et al, 2001 ). More recently the presence of a number of RAS components such as Ang I, Ang II, and Ang 1–7 was confirmed in mitochondria isolated from kidney cortex ( Wilson et al, 2016 , 2017 ). Mitochondrial angiotensins may be imported from the cytoplasm.…”

Megalin: a Novel Determinant of Renin-Angiotensin System Activity in the Kidney?

“…Obviously, endocytosis should then not result in angiotensinogen destruction. According to Wilson et al [58], internalized angiotensinogen traffics intact to the mitochondria in isolated proximal tubules. However, given the absence of intracellular angiotensin generation [4], the implications of this finding are unclear.…”