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 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min−1·mg protein−1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 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.