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.

Keywords: angiotensinogen; mitochondria; protein uptake; proximal tubules; renin-angiotensin system.

Fig. 1.

Uptake of ¹²⁵I-angiotensinogen in isolated proximal tubules. A: schematic of the ¹²⁵I-angiotensinogen uptake assay and subcellular fractionation. ¹²⁵I-angiotensinogen (20 nM) was incubated with isolated sheep renal proximal tubules (500 µg protein) for 2 h at 37°C. B: distribution of extracellular and intracellular ¹²⁵I-angiotensinogen. Data are mean ± SE expressed a %initial cpm; n = 4. Inset: subcellular fractionation revealed that 21 ± 4% of internalized ¹²⁵I-angiotensinogen associated with the mitochondrial (Mito) fraction, with additional labeling in the nucleus (Nuc; 60 ± 7%), endoplasmic reticulum (ER, 4 ± 0.5%), and cytosol (15 ± 4%). Data are means ± SE expressed as the %total intracellular cpm; n = 4.

Fig. 2.

Uptake of ¹²⁵I-angiotensinogen in sheep cortical mitochondria. A: schematic of mitochondrial ¹²⁵I-angiotensinogen uptake assay. B: increasing concentrations of ¹²⁵I-angiotensinogen result in a linear increase in mitochondrial uptake of ¹²⁵I-angiotensinogen. Nonspecific binding or uptake of ¹²⁵I-angiotensinogen where bovine serum albumin (BSA) was substituted for mitochondria in the uptake assay is included. Data are means ± SE; n = 3.

Fig. 3.

Rate of ¹²⁵I-angiotensinogen uptake is comparable between sheep and human mitochondria. A: sheep cortical mitochondria and human HK-2 mitochondria internalized ¹²⁵I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min⁻¹·mg protein⁻¹; n = 3). Inset: Purified sheep cortical (lanes 2–5) and HK-2 (lanes 6–9) mitochondria were fractionated by 10% PAGE-SDS and exposed to autoradiography film. A single protein band of 55 kDa corresponding to the molecular weight of the ¹²⁵I-angiotensinogen standard (Std, lane 1) was detected. B: unlabeled angiotensinogen (100nM) competed for ¹²⁵I-angiotensinogen uptake to a greater extent than human serum albumin (HAS; 100 nM) in the HK-2 mitochondria (60 ± 2 vs. 16 ± 13%). Data are means ± SE expressed as the %control uptake; P < 0.05; n = 3.

Fig. 4.

Characterization of ¹²⁵I-angiotensinogen import. Isolated HK-2 and sheep cortical mitochondria were exposed to the mitochondrial processing peptidase inhibitor o-phenanthroline (O-PHEN; 10 µM) or the mitochondrial membrane uncoupling agents carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 10 µM), valinomycin (VAL; 10 µM), and their combination (CCCP/VAL). OPHEN, CCCP, or VAL did not inhibit ¹²⁵I-angiotensinogen import. Data are means ± SE expressed as the %control uptake; P > 0.05, n = 3.

Fig. 5.

Uptake of ¹²⁵I-angiotensinogen is dependent on the outer mitochondrial membrane. A and B: purified sheep cortical (A) and HK-2 (B) mitochondria were exposed to the nonionic detergent digitonin (5 mg/ml) for various times to remove the outer mitochondrial membrane. Following the digitonin treatment, mitochondrial preparations were subjected to 10% SDS gel fractionation and immunoblotting with an antibody to VDAC. Digitonin treatment depleted the outer membrane marker VDAC in both sheep and human HK-2 mitochondria. C: digitonin treatment at 15 min significantly reduced ¹²⁵I-angiotensinogen uptake. Data are means ± SE; *P < 0.05 vs. control; n = 3.